Non-invasive energy upconversion methods and systems

ABSTRACT

Products, compositions, systems, and methods for modifying a target structure which mediates or is associated with a biological activity, including treatment of conditions, disorders, or diseases mediated by or associated with a target structure, such as a virus, cell, subcellular structure or extracellular structure. The methods may be performed in situ in a non-invasive manner by placing a nanoparticle having a metallic shell on at least a fraction of a surface in a vicinity of a target structure in a subject and applying an initiation energy to a subject thus producing an effect on or change to the target structure directly or via a modulation agent. The nanoparticle is configured, upon exposure to a first wavelength λ 1 , to generate a second wavelength λ 2  of radiation having a higher energy than the first wavelength λ 1 . The methods may further be performed by application of an initiation energy to a subject in situ to activate a pharmaceutical agent directly or via an energy modulation agent, optionally in the presence of one or more plasmonics active agents, thus producing an effect on or change to the target structure. Kits containing products or compositions formulated or configured and systems for use in practicing these methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/247,367, filed Aug. 25, 2016, now allowed, which is a continuation ofU.S. patent application Ser. No. 14/168,795 filed Jan. 30, 2014, nowU.S. Pat. No. 9,526,913, which is a continuation of U.S. patentapplication Ser. No. 12/764,184 filed Apr. 21, 2010, now U.S. Pat. No.9,302,116, and is related to U.S. patent application Ser. No.11/935,655, filed Nov. 6, 2007 and Ser. No. 12/059,484, filed Mar. 31,2008; U.S. patent application Ser. No. 12/389,946, filed Feb. 20, 2009;U.S. patent application Ser. No. 12/417,779, filed Apr. 3, 2009, U.S.patent application Ser. No. 12/725,108, filed Mar. 16, 2010, andprovisional patent applications 61/161,328, filed Mar. 18, 2009, and61/259,940, filed Nov. 10, 2009; the entire contents of each of whichare hereby incorporated by reference. This application is also relatedto and claims priority from provisional patent application 61/171,152,filed Apr. 21, 2009; the entire contents of each of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION Field of Invention

The present invention relates to methods and systems that can beperformed using energy up-conversion in non-invasive or minimallyinvasive techniques.

Discussion of the Background

Presently, light (i.e., electromagnetic radiation from the radiofrequency through the visible to the X-ray wavelength range) is used ina number of industrial, communication, electronic, and pharmaceuticalprocesses. Light in the infrared and visible range is typicallygenerated from an electrical energy source which for example eitherheats a material to extremely high temperatures where black bodyemission occurs (as in an incandescent lamp). Light in the visible andultraviolet range is typically generated by heating a gas to anelectrical discharge where transitions from one electronic state of thegas atom or molecule occurs with the emission of light. There are alsosemiconductor based light sources (as in light emitting diodes andsemiconducting lasers) where electrons/holes in a material recombine toproduce light emission.

With the development of ultraviolet sources, ultraviolet radiation isbeing increasingly utilized for industrial, chemical, and pharmaceuticalpurposes. For example, UV light is known to sterilize media and islInown to drive a number of photo-activated chemical processes such asthe cross-linking of polymers in adhesives or coatings. Typically,ultraviolet sources use gas discharge lamps to generate emitted light inthe ultraviolet range. The emitted light is then optically filtered toremove many of not all of the non-ultraviolet frequencies. Ultravioletlight can also be produced in semiconductor phosphors from theexcitation of these phosphors from high energy sources such as, forexample, X-ray irradiation.

With the development of infrared radiation sources, infrared radiationis being increasingly utilized for communications and signalingpurposes. Typically, infrared sources use broad spectrum light sourcesreferred to as glowbars to generate a broad spectrum of light centeredin the infrared range or use lasers to emit very specific infraredwavelengths. For the broad band sources, the emitted light is opticallyfiltered to remove many of not all of the non-infrared frequencies.

It is generally desirable to have devices, materials, and capabilitiesto convert light from one frequency range to another. Down conversionhas been one way to convert higher energy light to lower energy, as usedin the phosphors noted above. Up conversion has also been shown wherelower energy light is converted to higher energy light. Typically, thisprocess is a multi-photon absorption process where two or more photonsare used to promote an excited electronic state in a host medium whichin turn radiates at a wavelength of light that has a higher energy thanthe energy of the incident light which promoted the multi-photonabsorption process. Both down conversion and up conversion have beenstudied and documented in the past.

Indeed, workers have studied the phenomenon of photoluminescence, whichis the ability of certain solids, known as phosphors, to emit light whendriven or charged by an external energy source. Many well-knownphosphors are triggered by high-energy electrons or photons and emitphotons of lower energy. However, there is another type of phosphorwhich can store energy for long periods of time in certain energystates. Relaxation from these energy states at a later time can bestimulated by less energetic photons. Relaxation from these energystates results in photon emission. The effect of this phenomenon is thatenergy is stored in the form of trapped electron-hole pairs for lateruse. Materials which exhibit this phenomenon will be referred to aselectron trapping, or electron trapping phosphors, and materials inwhich emission of light is activated by infrared lumination are calledinfrared phosphors.

It has been recognized recently that certain infrared phosphors canactually operate at high speeds and are capable of converting pulsedinfrared light to the visible range (violet through red). This“upconversion” occurs at the expense of the original chargingilluminating light and can actually exhibit optical gain. It has beenobserved that phosphorescence can continue for as long as several daysbefore a new short recharge is required.

Up conversion and down conversion of electromagnetic radiations are veryrelevant to various industrials fields. Photo-activated chemicalreactions find broad use in the industry from catalyzing reactions toBio-modulation of therapeutic agents. However, UV radiation suffers froma lack of depth of penetration in matter especially biological media,polymers and most solids). For this reason, UV based photo-initiation islimited by direct line of site which prevents volumetric applications.

UV has been limited to reactions taking place on the outer surfaces ofmaterials may they be solids or liquids; organic or inorganic;biological organs, living tissues and composites thereof, structuralcomposites, materials residing inside chemical tanks/reactors for foodprocessing or hydrocarbon chains fractionation (to name a few examples).

Photobiomodulation

Photobiomodulation also known as low level laser therapy (LLLT), coldlaser therapy, and laser biostimulation, is an emerging medical andveterinary technique in which exposure to low-level laser light canstimulate or inhibit cellular function leading to beneficial clinicaleffects. The “best” combination of wavelength, intensity, duration andtreatment interval is complex and sometimes controversial with differentdiseases, injuries and dysfunctions needing different treatmentparameters and techniques.

Certain wavelengths of light at certain intensities (delivered by laser,LED or another monochromatic source) will, for example, aid tissueregeneration, resolve inflammation, relieve pain and boost the immunesystem. The exact mechanism is still being explored and debated but itis agreed that the mechanism is photochemical rather than heat-related.Observed biological and physiological effects include changes in cellmembrane permeability, and up-regulation and down-regulation ofadenosine triphosphate and nitric oxide.

All light-induced biological effects depend on the parameters of theirradiation (wavelength, dose, intensity, irradiation time, depth of atarget cell, and continuous wave or pulsed mode, pulse parameters),(See, e.g., Karu I T, Low-Power Laser Therapy”, in Biomedical PhotonicsHandbook, Vo-Dinh T. Ed., CRC Press, Boca Raton, Fla., pp, 48-1 to48-25, (2003)). Laser average power is typically in the range of 1-500mW; some high peak power, short pulse width devices are in the range of1-100 W with typically 200 as pulse widths. The average beam irradiancethen is typically 10 mW/cm²-5 W/cm². The wavelength is typically in therange 600-1000 nm. The red-to-near infrared (NIR) region is preferredfor photobiomodulation. Other wavelengths may be also used, e.g., UVlight for neurons and green light for prostate tissue. Maximumbiological responses are occurring when irradiated at 620, 680, 760, and820-830 nm. (Karu T I, et al, (1998). The Science of Low Power LaserTherapy, Gordon and Breach Sci. Publ., London). Large volumes andrelatively deeper layers of tissues can be successfully irradiated bylaser only (e.g., inner and middle ear diseases, injured siatic oroptical nerves, inflammations). The LEDs are used for irradiation ofsurface injuries.

A photoacceptor must first absorb the light used for the irradiation.After promotion of electronically excited states, primary moleculeprocesses from these states can lead to a measurable biological effect(via secondary biochemical reaction, or photosignal transductioncascade, or cellular signaling) at the cellular level. A photoacceptorfor eukaryotic cells in red-to-NIR region is believed to be the terminalenzyme of the respiratory chain cytochrome c oxidase located in cellmitochondrion. In the violet-to blue spectra region, flavoprotein (e.g.,NADHdehydrogenase in the beginning of the respiratory chain) is alsoamong the photoacceptors.

Clinical applications of photobiomodulation include, for example,treating soft tissue and bone injuries, chronic pain, wound healing andnerve and sensory regeneration/restoration, and possibly even resolvingviral and bacterial infections, treating neurological and phychiatricdiseases (e.g., epilepsy and Parkinson's disease) (e.g., Zhang F., etal., Nature, 446:617-9 (Apr. 5, 2007; Han X., et al., PloS ONE,2(3):e299 (Mar. 21, 2007); Arany P R, et al., Wound Repair Regen.,15(6):866-74 (2007); Lopes C B, et al., Photomed. Laser Surg.,25(2):96-101 (2007)). One clinical application showing great promise isthe treatment of inflammation, where the anti-inflammatory effect oflocation-and-dose-specific laser irradiation produces similar outcomesas NSAIDs, but without the potentially harmful side-effects (Bjordal JM, Couppé C, Chow R T, Tunér J, Ljunggren E A (2003). “A systematicreview of low level laser therapy with location-specific doses for painfrom chronic joint disorders”. The Australian journal of physiotherapy49(2):107-16).

An NIR light treatment can prevent cell death (apoptosis) in culturedneurons (brain) cells (Wong-Reiley M T, et al., JBC, 280(6):4761-71(2005)). Specific wavelengths of light can promote cellularproliferation to the activation of mitochondria, the energy-producingorganelles within the cell via cytochrome c oxidase. An NIR treatmentcan augment mitochondrial function and stimulate antioxidant protectivepathways. The evidence that the NIR treatment can augment mitochondrialfunction and stimulate antioxidant protective pathways comes fromphotobiomodulation experiments carried out using a laboratory model ofParkinson's disease (PD) (cultures of human dopaminergic neuronal cells)(Whelan H., et. al., SPIE, Newsroom, pages 1-3 (2008)).

It has also been shown that light has both inductive and inhibitoryeffect on cell growth and division in a red tide flagellate, Chattonellaantique (Nemote Y., Plant and Cell Physiol., 26(4):669-674 (1985)).

When the excitable cells (e.g., neurons, cardiomyocites) are irradiatedwith monochromatic visible light, the photoacceptors are also believedto be components of respiratory chain. It is clear from experimentaldata (Karu, T. I., (2002). Low-power laser therapy. In: CRC BiomedicalPhotonics Handbook, T. Vo-Dinh, Editor-in-Chief, CRC Press, Boca Raton(USA)) that irradiation can cause physiological and morphologicalchanges in nonpigmental excitable cells via absorption in mitochondria.Later, similar irradiation experiments were performed with neurons inconnection with low-power laser therapy. It was shown in 80's that He—Nelaser radiation alters the firing pattern of nerves; it was also foundthat transcutaneous irradiation with HeNe laser mimicked the effect ofperipheral stimulation of a behavioral reflex. These findings were foundto be connected with pain therapy (Karu T I, et al., (2002)).

When photoacceptors absorb photons, electronic excitation followed byphotochemical reactions occurring from lower excitation states (firstsinglet and triplet) takes place. It is also known that electronicexcitation of absorbing centers alters their redox properties. Untilyet, five primary reactions have been discussed in literature (Karu T I,et al., (2002)). Two of them are connected with alteration of redoxproperties and two mechanisms involve generation of reactive oxygenspecies (ROE). Also, induction of local transient (very short time)heating of absorbing chromophores is possible. Details of thesemechanisms can be found in (Karu T I, et. al., (2002); Karu T I, et al.,(1998). The Science of Low Power Laser Therapy. Gordon and Breach Sci.Publ., London).

Photobiological action via activation of respiratory chain is believedto be a general mechanism occurring in cells. Crucial events of thistype of cell metabolism activation are occurring due to a shift ofcellular redox potential into more oxidized direction as well as due toATP extrasynthesis. Susceptibility to irradiation and capability foractivation depend on physiological status of irradiated cells: thecells, which overall redox potential is shifted to more reduced state(example: some pathological conditions) are more sensitive to theirradiation. The specificity of final photobiological response isdetermined not at the level of primary reactions in the respiratorychain but at the transcription level during cellular signaling cascades.In some cells, only partial activation of cell metabolism happens bythis mechanism (example: redox priming of lymphocytes).

Far red and NIR radiation have been shown to promote wound healing,e.g., infected, ischemic, and hypoxic wounds (Wong-Reley, W T T, JBC,280(6):4761-4771 (2005)). Red-to-NIR radiation also protects the retinaagainst the toxic actions of methanol-derived formic acid in a rodentmodel of methanol toxicity and may enhance recovery from retinal injuryand other ocular diseases in which mitochondrial dysfunction ispostulated to play a role (Eells J T., PNAS, 100(6):3439-44 (2003)).Another clinical application of photobiomodulation is repair of soft andbone tissues by IR laser irradiation (Martinez M E, et al., Laser inMed. Sci., 2007). Invasive laser assisted liposuction is a recentlydeveloped method, wherein a laser fiber is introduced through a tubeinto the skin and directly to the fat cells causing the cells to raptureand drain away as liquid (Kim K H, Dermatol. Surg., 32(2):241-48(2006)). Tissue around the area is coagulated. Yet, another applicationof photobiomodulation is a non-surgical varicose vein treatment (anendovenous laser therapy), wherein a laser is threaded through anincision and the full length of the varicose vein (Kim H S, J. Vasc.Interv. Radiol., 18(6):811 (2007)). When the laser is slowly withdrawn,heat is applied to the vein walls, causing the vein to permanently closeand disappear.

Technological advances such as laser have redefined the surgicaltreatment of enlarged prostate. The green light laser is a laser thatvaporizes and removes the enlarged prostate tissue (Heinrich E., Eur.Urol., 52(6):1632-7 (2007)). The significance of the color of the laserlight (green) is that this results in absorption by hemoglobin which iscontained within red blood cells and not absorbed by water. Theprocedure may also be known as laser prostatectomy or laserTransurethral resection of the prostate (TURP). The technique involvespainting the enlarged prostate with the laser until the capsule of theprostate is reached. By relieving this portion of the prostate, patientsare able to void much easier through a wide-open channel in theprostate. The procedure needs to be performed under general or spinalanesthesia. An advantage of the procedure is that even patients takingblood thinners (e.g., aspirin to prevent stroke) can be treated becausethere is less bleeding compared to a traditional surgery.

Yet, another area of application of photobiomodulation is a directcontrol of brain cell activity with light. The technique is based uponNIR spectroscopy and is simpler to use and less expensive than othermethods such as functional magnetic resonance imaging and positronemission tomography.

Whenever a region of the brain is activated, that part of the brain usesmore oxygen. This technique works by measuring the blood flow and oxygenconsumption in the brain. The light emitted by NIR laser diodes iscarried through optical fibers to a person's head. The light penetratesthe skull where it assesses the brain's oxygen level and blood volume.The scattered light is then collected by optical fibers, sent todetectors and analyzed by a computer. By examining how much of the lightis scattered and how much is absorbed, portions of the brain and extractinformation about brain activity can be mapped. By measuring thescattering, it is determined where the neurons are firing. This meansthat scientists can simultaneously detect both blood profusion andneural activity. The technique could be used in many diagnostic,prognostic and clinical applications. For example, it could be used tofind hematomas in children, to study blood flow in the brain duringsleep apnea, and to monitor recovering stroke patients on a daily, oreven hourly, basis (that would be impractical to do with MRI). Tovalidate the technique, hemoglobin oxygen concentrations in the brainobtained simultaneously by NIR spectroscopy and by functional MRI, thecurrent “gold standard” in brain studies, was compared. Both methodswere used to generate functional maps of the brain's motor cortex duringa periodic sequence of stimulation by finger motion and rest. Spatialcongruence between the hemoglobin signal and the MRI signal in the motorcortex related to finger movement was demonstrated. The researchers alsodemonstrated collocation between hemoglobin oxygen levels and changes inscattering due to brain activities. The changes in scattering associatedwith fast neuron signals came from exactly the same locations.

A low-intensity laser light-oxygen cancer therapy is another applicationof photobiomodulation. The light-oxygen effect (LOE), which involvesactivation of or damage to biosystems by optical radiation at lowoptical doses by direct photoexcitation of molecular oxygen dissolved ina biosystem so that it is converted to the singlet state, i.e., byphotogeneration of molecular singlet oxygen from O₂ dissolved in cells,similar to photodynamic effect (Zakharov S D, et al., QuantumElectronics, 29(12): 1031-53 (1999)). It was shown that the He—Ne laserradiation destroys tumor cells in the presence or absence of thephotosensitiser. The LOE can be activated by small optical doses, whichare 4-5 orders of magnitude lower that those found if a comparison ismade with the familiar analogue in the form of the photodynamic effect(PDE).

Photobiostimulation Using “Caged” Molecules and Light-Sensitive Proteins

This type of photobiomodulation methods fall into two generalcategories: one set of methods uses light to uncage a compound that thenbecomes biochemically active, binding to a downstream effector. Forexample, this method involves applying “caged” chemicals to a sample andthen using light to open the cage to invoke a reaction. Modifiedglutamate is useful for finding excitatory connections between neurons,since the uncaged glutamate mimics the natural synaptic activity of oneneuron impinging upon another. This method is used for elucidation ofneuron functions and imaging in brain slices using, for example,two-photon glutamine uncageing (Harvey C D, et al., Nature,450:1195-1202 (2007); Eder M, et al., Rev. Neurosci., 15:167-183(2004)). Other signaling molecules can be released by UV lightstimulation, e.g., GABA, secondary messengers (e.g., Ca²⁺ and Mg²⁺),carbachol, capsaicin, and ATP (Zhang F., et al., 2006).

The other major photostimulation method is the use of light to activatea light-sensitive protein such as rhodopsin (ChR2), which can thenexcite the cell expressing the opsin.

It has been shown that channelrhodopsin-2, a monolithic proteincontaining a light sensor and a cation channel, provides electricalstimulation of appropriate speed and magnitude to activate neuronalspike firing. Recently, photoinhibition, the inhibition of neuralactivity with light, has become feasible with the application ofmolecules such as the light-activated chloride pump halorhodopsin toneural control. Together, blue-light activated channelrhodopsin-2 andthe yellow light-activated chloride pump halorhodopsin enablemultiple-color, optical activation and silencing of neural activity.

ChR2 photostimulaiton involves genetic targeting ChR2 to neurons andlight pulsing the neurons expressing ChR2 protein. The experiments havebeen conducted in vitro and in vivo in mice by in vivo deep-brainphotostimulaiton using optical fibers to deliver light into the lateralhypothalamus (Adamantidis A R, et al., Nature 450:420-425 (2007)).Genetic targeting of ChR2 allows exclusive stimulation of definedcellular subsets and avoids the need for addition of the cagedglutamate, facilitating photostimulation in vivo (Wang H., et al., PNAS,104(19):8143-48 (2007)). ChR2 photostimulation has been used forrestoring visual activity in mice with impaired vision, to evokebehavioral responses in worms and flies (Wang H., et al., 2007). Therobust associative learning induced by ChR2-assisted photostimulaiton inmice opens the door to study the circuit basis of perception andcognition in vivo (Huber D., et al., 2007). This kind of neuronaltargeting and stimulation might have clinical application, e.g., deepbrain stimulation to treat Parkinson's disease and other disorders,controlling behavioral, perceptional and cognitive characteristics, andfor imaging and studying how the brain works (Zhang F., et al., NatureMethods, 3(10):785-792 (2006); Wong-Riley M T., et al., JBC,280(6):4761-4771 (2005)).

Another gene, chloride pump (NpHR), which is borrowed from a microbecalled an archaebacterium, can make neurons less active in the presenceof yellow light. Combined, the two genes ChR2 and NpHR can now makeneurons obey pulses of light like drivers obey a traffic signal: Bluemeans “go” (emit a signal), and yellow means “stop” (don't emit).

Light-sensitive proteins can be introduced into cells or live subjectsvia a number of techniques including electroporation, DNAmicroinjection, viral delivery, liposomal transfection andcalcium-phosphate precipitation.

A third photostimulation technique is chemical modification of ionchannels and receptors to render them light-responsive. Some of the mostfundamental signaling mechanisms in a cell involve the release anduptake of Ca²⁺ ions. Ca²⁺ is involved in controlling fertilization,differentiation, proliferation, apoptosis, synaptic plasticity, memory,and developing axons. It has been shown that Ca²⁺ waves can be inducedby UV irradiation (single-photon absorption) and NIR irradiation(two-photon absorption) by releasing caged Ca²⁺, an extracellularpurinergic messenger InsP3 (Bract K., et al., Cell Calcium, 33:37-48(2003)), or ion channel ligands (Zhang F., et al., 2006).

Directly controlling a brain cell activity with light is a novel meansfor experimenting with neural circuits and could lead to therapies forsome disorders. This accomplishment is a step toward the goal of mappingneural circuit dynamics on a millisecond timescale to see if impairmentsin these dynamics underlie severe psychiatric symptoms. Knowing theeffects that different neurons have could ultimately help researchersfigure out the workings of healthy and unhealthy brain circuits. If useof the technique can show that altered activity in a particular kind ofneuron underlies symptoms, for example, this insight will allowdevelopment of targeted genetic or pharmaceutical treatments to fixthose neurons. Conceivably, direct control of neuronal activity withlight could someday become a therapy in itself.

In living organisms, scientists were able to cause worms, C. elegans, tostop swimming while their genetically altered motor neurons were exposedto pulses of yellow light intensified through a microscope. In someexperiments, exposure to blue light caused the worms to wiggle in waysthey weren't moving while unperturbed. When the lights were turned off,the worms resumed their normal behavior.

Meanwhile, in experiments in living brain tissues extracted from mice,the researchers were able to use the technique to cause neurons tosignal or stop on the millisecond timescale, just as they do naturally,Other experiments showed that cells appear to suffer no ill effects fromexposure to the light. They resume their normal function once theexposure ends.

The most direct application of an optical neuron control isexperimenting with neural circuits to determine why unhealthy ones failand how healthy ones work.

In patients with Parkinson's disease, for example, researchers haveshown that electrical “deep brain stimulation” of cells can helppatients, but they don't know precisely why. By allowing researchers toselectively stimulate or dampen different neurons in the brain, thelight stimulation techniques could help in determining which particularneurons are benefiting from deep brain stimulation. That could lead tomaking the electrical treatment, which has some unwanted side effects,more targeted.

Another potential application is experimenting with simulating neuralcommunications. Because neurons communicate by generating patterns ofsignals-sometimes on and sometimes off like the 0s and 1s of binarycomputer code-flashing blue and yellow lights in these patterns couldcompel neurons to emit messages that correspond to real neuralinstructions. In the future, this could allow researchers to test andtune sophisticated neuron behaviors. Much farther down the road, theability to artificially stimulate neural signals, such as movementinstructions, could allow doctors to bridge blockages in damaged spinalcolumns, perhaps restoring some function to the limbs of paralyzedpatients.

Finally, the technique could be useful in teasing out the largelyunknown functioning of healthy brains.

Problems with LLLT, Cold Laser Therapy, and Laser Biostimulation

The laser systems currently used for biostimulation do not allowperforming photobiomodulation in a region deep within thick tissuewithout a surgical invasion. Laser therapy is mostly conducted insurface or near surface target cells and tissue because penetration ofUV and red-to-NIR radiation used for photobiomodulation andphotobiostimulaiton is no more than a few centimeters beneath thesurface of the skin. In addition, imaging and stimulation of brain cellsis mainly possible in thin brain slices, or a thin monolayer orsuspension of cells. For deeper tissue laser therapy in situ, a subjectundergoes various invasive surgical procedures, e.g., invasive insertionof a fiber via incisions into a fat layer or veins, implanting aradiation source in deep tissue, or implanting a glass window above thebarrel cortex (Huber D., et al., Nature, 451:61-66 (2007)). It isfurther well recognized that another problem associated with theexisting methods of photobiomodulation is in differentiation of normalcells from target cells.

Traditional Methods in Phototherapy

Photopheresis, also known as extracorporeal photochemotherapy (ECP),involves the removal and reinfusion of autologous blood after the whiteblood cell portion was collected, treated extracorporeally with aphotosensitizing drug and irradiated with ultraviolet A light. Whenreinfused into the patient's body, lymphocytes bound to thephotoactivated drug act like a vaccine to alert the immune system todestroy any similar T-cells circulating in the blood. Photopheresis hasbeen successfully used for treatment of cell proliferation disorders.Exemplary cell proliferation disorders may include, but are not limitedto, cancer, bacterial infection, immune rejection response of organtransplant, solid tumors, viral infection, autoimmune disorders (such asarthritis, lupus, inflammatory bowel disease, Sjogrens syndrome,multiple sclerosis) or a combination thereof, as well as aplasticconditions wherein cell proliferation is low relative to healthy cells,such as aplastic anemia. Of these, cancer is perhaps the most wellknown.

Excellent results have been observed since its initial approval by theFDA in 1988. Photopheresis is currently approved for the treatment ofrefractory cutaneous T-cell lymphoma.

Extracorporeal photopheresis is a leukapheresis-based immunomodulatorytherapy that has been approved by the US Food and Drug Administrationfor the treatment of cutaneous T-cell lymphoma (CTCL). ECP, also knownas extracorporeal photochemotherapy, is performed at more than 150centers worldwide for multiple indications. Long-term follow-up data areavailable from many investigators that indicate ECP produces diseaseremission and improved survival for CTCL patients. In addition to CTCL,ECP has been shown to have efficacy in the treatment of other T-cellmediated disorders, including chronic graft versus host disease (GVHD)and solid organ transplant rejection. ECP use for the treatment ofautoimmune disease, such as systemic sclerosis and rheumatoid arthritis,is also being explored.

ECP is generally performed using the UVAR XTS Photopheresis Systemdeveloped by Therakos, Inc (Exton, Pa.). The process is performedthrough one intravenous access port and has 3 basic stages: (1)leukapheresis, (2) photoactivation, and (3) reinfusion, and takes 3-4hours to complete. A typical treatment session would resemble thefollowing sequence of events:

(1) One 16-gauge peripheral intravenous line or central venous access isestablished in the patient;

(2) Blood (225 mL) is passed through 3 cycles of leukapheresis, or 125mL of blood is passed through 6 cycles, depending on the patient'shematocrit value and body size. At the end of each leukapheresis cycle,the red blood cells and plasma are returned to the patient;

(3) The collected WBCs (including approximately 5% of the peripheralblood mononuclear cells) are mixed with heparin, saline, and8-methoxypsoralen (8-MOP), which intercalates into the DNA of thelymphocytes upon exposure to UVA light and makes them more susceptibleto apoptosis when exposed to UVA radiation;

(4) The mixture is passed as a 1-mm film through a sterile cassettesurrounded by UVA bulbs, resulting in an average UVA exposure of 2J/cm²; and

(5) The treated WBC mixture is returned to the patient.

Over the past 20 years, on-going research has explored the mechanism ofaction of ECP. The combination of 8-MOP and UVA radiation causesapoptosis of the treated T cells and may cause preferential apoptosis ofactivated or abnormal T cells, thus targeting the pathogenic cells ofCTCL or GVHD. However, given that only a small percentage of the body'slymphocytes are treated, this seems unlikely to be the only mechanism ofaction.

Other evidence suggests that ECP also induces monocytes to differentiateinto dendritic cells capable of phagocytosing and processing theapoptotic T-cell antigens. When these activated dendritic cells arereinfused into the systemic circulation, they may cause a systemiccytotoxic CD8⁺ T-lymphocyte-mediated immune response to the processedapoptotic T-cell antigens.

Finally, animal studies indicate that photopheresis may induceantigen-specific regulatory T cells, which may lead to suppression ofallograft rejection or GVHD.

However, there are still many limitations to ECP. For example, ECPrequires patient to be connected to a machine for hours per treatment.It requires establishing peripheral intravenous line or central venousaccess, which may be difficult to do in certain disease states such assystemic sclerosis or arthritis. There is also a risk of infection atthe venous or central line site, or in the central line catheter.Further, it requires removing typically several hundred milliliters ofwhole blood from the patient, hence, the treatment is limited topatients who has sufficiently large initial volume of blood to bewithdrawn. The American Association of Blood Blanks recommend a limit ofextracorporeal volume to 15% of the patient's whole body blood volume.Therefore, the size of the volume that can be treated generally has tobe at least 40 kg or more. Risk of contracting blood-born pathogen(Hepatitis, HIV, etc.) due to exposure to contaminated operating systemis also a concern.

Alternatively, a patient can be treated in vivo with a photosensitiveagent followed by the withdrawal of a sample from the patient, treatmentwith UV radiation in vitro (ex vivo), and reinjecting the patient withthe treated sample. This method is known for producing an autovaccine. Amethod of treating a patient with a photosensitive agent, exposing thepatient to an energy source and generating an autovaccine effect whereinall steps are conducted in vivo has not been described. See WO03/049801, U.S. Pat. Nos. 6,569,467; 6,204,058; 5,980,954; 6,669,965;4,838,852; 7,045,124, and 6,849,058. Moreover, the side effects ofextracorporeal photopheresis are well known and include nausea,vomiting, cutaneous erythema, hypersensitivity to sunlight, andsecondary hematologic malignancy. Researchers are attempting to usephotopheresis in experimental treatments for patients with cardiac,pulmonary and renal allograft rejection; autoimmune diseases, andulcerative colitis.

Phototherapy is a relatively new light-based treatment, which hasrecently been approved by the United States Food & Drug Administration(FDA) for the treatment of both early and late-stage lung cancer. Fortumors occurring deep in tissue, second generation sensitizers, whichhave absorbance in the NIR region, such as porphyrin-based systems [R.K. Pandey, “Synthetic Strategies in designing Porphyrin-BasedPhotosensitizers”, in Biomedical Photonics Handbook, Vo-Dinh T., Ed.,CRC Press, Boca Raton Fla. (2003)], chlorines, phthalocyanine, andnaphthalocyanine have been investigated.

There are two main types of reactions in phototherapy:

-   -   (1) Type I reactions involve electrons and hydrogen atoms, which        are transferred between photo-active molecules (also called        photosensitizers) and substrates or solvent molecules. Oxygen        may participate in subsequent reactions: e.g., psoralens in        photopheresis and PUVA.    -   (2) Type II reactions involve singlet oxygen formation by energy        transfer from PA molecules in the lowest triplet state to oxygen        in the ground state: e.g., photodynamic therapy (PDT)

Photodynamic therapy (PDT) is a treatment modality that uses aphotosensitizing agent and laser light to kill cells. PDT is arelatively new light-based treatment, which has recently been approvedby the United States Food & Drug Administration (FDA) for the treatmentof both early and late-stage lung cancer. Other countries have approvedPDT for treatment of various cancers as well. Unlike chemotherapy,radiation, and surgery, PDT is useful in treating all cell types,whether small cell or non-small cell carcinoma. PDT involves treatmentof diseases such as cancer using light action on a special photoactiveclass of drugs, by photodynamic action in vivo to destroy or modifytissue [Dougherty T. J. and Levy J. G., “Photodynamic Therapy andClinical Applications”, in Biomedical Photonics Handbook, Vo-Dinh T.,Ed., CRC Press, Boca Raton Fla. (2003)]. PDT, which was originallydeveloped for treatment of various cancers, has now been used to includetreatment of pre-cancerous conditions, e.g. actinic keratoses,high-grade dysplasia in Barrett's esophagus, and non-cancerousconditions, e.g. various eye diseases, e.g. age related maculardegeneration (AMD). Photodynamic therapy (PDT) is approved forcommercialization worldwide both for various cancers (lung, esophagus)and for AMD.

The PDT process requires three elements: (1) a PA drug (i.e.,photosensitizer), (2) light that can excite the photosensitizer and (3)endogenous oxygen. The putative cytotoxic agent is singlet oxygen, anelectronically excited state of ground state triplet oxygen formedaccording to the Type II photochemical process, as follows.

PA+hv→¹PA*(S) Excitation

¹PA*(S)→³PA*(T) Intersystem crossing for singlet to triplet state

³PA*(T)+O₂→¹O*₂+PA Energy transfer from the drug to singlet oxygen

where PA=photo-active drug at the ground state; ¹PA*(S)=excited singletstate;

³PA*(T)=excited triplet state; ¹O*₂=singlet excited state of oxygen

Because the triplet state has a relatively long lifetime (μsec toseconds) only photosensitizers that undergo efficient intersystemcrossing to the excited triplet state will have sufficient time forcollision with oxygen in order to produce singlet oxygen. The energydifference between ground state and singlet oxygen is 94.2 kJ/mol andcorresponds to a transition in the near-infrared at ˜1270 nm. Most PAphotosensitizers in clinical use have triplet quantum yields in therange of 40-60% with the singlet oxygen yield being slightly lower.Competing processes include loss of energy by deactivation to groundstate by fluorescence or internal conversion (loss of energy to theenvironment or surrounding medium).

However, while a high yield of singlet oxygen is desirable it is by nomeans sufficient for a photosensitizer to be clinically useful.Pharmacokinetics, pharmacodynamics, stability in vivo and acceptabletoxicity play critical roles as well [Henderson B W, Gollnick S O,“Mechanistic Principles of Photodynamic Therapy”, in BiomedicalPhotonics Handbook, Vo-Dinh T., Ed., CRC Press, Boca Raton Fla. (2003)].For example, it is desirable to have relatively selective uptake in thetumor or other tissue being treated relative to the normal tissue thatnecessarily will be exposed to the exciting light as well.Pharmacodynamic issues such as the subcellular localization of thephotosensitizer may be important as certain organelles appear to be moresensitive to PDT damage than others (e.g. the mitochondria). Toxicitycan become an issue if high doses of photosensitizer are necessary inorder to obtain a complete response to treatment. An important mechanismassociated with PDT drug activity involves apoptosis in cells. Uponabsorption of light, the photosensitiser (PS) initiates chemicalreactions that lead to the direct or indirect production of cytotoxicspecies such as radicals and singlet oxygen. The reaction of thecytotoxic species with subcellular organelles and macromolecules(proteins, DNA, etc) lead to apoptosis and/or necrosis of the cellshosting the PDT drug. The preferential accumulation of PDT drugmolecules in cancer cells combined with the localized delivery of lightto the tumor, results in the selective destruction of the cancerouslesion. Compared to other traditional anticancer therapies, PDT does notinvolve generalized destruction of healthy cells. In addition to directcell killing, PDT can also act on the vasculature, reducing blood flowto the tumor causing its necrosis. In particular cases it can be used asa less invasive alternative to surgery.

There are several chemical species used for PDT includingporphyrin-based sensitizers. A purified hematoporphyrin derivative,Photofrin®, has received approval of the US Food and DrugAdministration. Porphyrins are generally used for tumors on or justunder the skin or on the lining of internal organs or cavities becausetheses drug molecules absorbs light shorter than 640 nm in wavelength.For tumors occurring deep in tissue, second generation sensitizers,which have absorbance in the NIR region, such as porphyrin-based systems[R. K. Pandey, “Synthetic Strategies in designing Porphyrin-BasedPhotosensitizers”, in Biomedical Photonics Handbook, Vo-Dinh T., Ed.,CRC Press, Boca Raton Fla. (2003)], chlorines, phthalocyanine, andnaphthalocyanine have been investigated.

PDT retains several photosensitizers in tumors for a longer time than innormal tissues, thus offering potential improvement in treatmentselectivity. See Comer C., “Determination of [3H]- and [14C]hematoporphyrin derivative distribution in malignant and normal tissue,”Cancer Res 1979, 3 9: 146-151; Young S W, et al., “Lutetium texaphyrin(PCI-0123) a near-infrared, water-soluble photosensitizer,” PhotochemPhotobiol 1996, 63:892-897; and Berenbaum M C, et al.,“Meso-Tetra(hydroxyphenyl)porphyrins, a new class of potent tumorphotosensitisers with favorable selectivity,” Br J Cancer 1986,54:717-725. Photodynamic therapy uses light of a specific wavelength toactivate the photosensitizing agent. Various light sources have beendeveloped for PDT, which include dye lasers and diode lasers. Lightgenerated by lasers can be coupled to optical fibers that allow thelight to be transmitted to the desired site. See Pass 1-11,“Photodynamic therapy in oncology: mechanisms and clinical use,” J NatlCancer Inst 1993, 85:443-456. According to researchers, the cytotoxiceffect of PDT is the result of photooxidation reactions, as disclosed inFoote C S, “Mechanisms of photooxygenation,” Proa Clin Biol Res 1984,170:3-18. Light causes excitation of the photosensitizer, in thepresence of oxygen, to produce various toxic species, such as singletoxygen and hydroxyl radicals. It is not clear that direct damage to DNAis a major effect; therefore, this may indicate that photoactivation ofDNA crosslinking is not stimulated efficiently.

Furthermore, when laser light is administered via external illuminationof tissue surfaces, the treatment effect of PDT is confined to a fewmillimeters (i.e. superficial). The reason for this superficiallimitation is mainly the limited penetration of the visible light usedto activate the photosensitizer. Thus, PDT is used to treat the surfacesof critical organs, such as lungs or intra-abdominal organs, withoutdamage to the underlying structures. However, even these treatmentsrequire significantly invasive techniques to treat the surface of theaffected organs. Clinical situations use the procedure in conjunctionwith surgical debulking to destroy remnants of microscopic or minimalgross disease. It is possible that the laser light and small amount ofremaining microscopic and minimal gross disease results in too little orhighly damaged structures. Pre-clinical data show that some immuneresponse is generated, but clinical trials have reported no auto vaccineeffect similar to that produced by extracorporeal photopheresis inclinical conditions. Instead, the immune response appears to be vigorousonly under limited conditions and only for a limited duration.

PDT retains several photosensitizers in tumors for a longer time than innormal tissues, thus offering potential improvement in treatmentselectivity. See Corner C., “Determination of [3H]- and [14C]hematoporphyrin derivative distribution in malignant and normal tissue,”Cancer Res 1979, 39: 146-151; Young S W, et al., “Lutetium texaphyrin(PCI-0123) a near-infrared, water-soluble photosensitizer,” PhotochemPhotobiol 1996, 63:892-897; and Berenbaum M C, et al.,“Meso-Tetra(hydroxyphenyl)porphyrins, a new class of potent tumorphotosensitisers with favorable selectivity,” Br J Cancer 1986,54:717-725. Photodynamic therapy uses light of a specific wavelength toactivate the photosensitizing agent. Various light sources have beendeveloped for PDT that include dye lasers and diode lasers. Lightgenerated by lasers can be coupled to optical fibers that allow thelight to be transmitted to the desired site. See Pass 1-11,“Photodynamic therapy in oncology: mechanisms and clinical use,” J NatlCancer Inst 1993, 85:443-456. According to researchers, the cytotoxiceffect of PDT is the result of photooxidation reactions, as disclosed inFoote C S, “Mechanisms of photooxygenation,” Proa Clin Biol Res 1984,170:3-18. Light causes excitation of the photosensitizer, in thepresence of oxygen, to produce various toxic species, such as singletoxygen and hydroxyl radicals. It is not clear that direct damage to DNAis a major effect; therefore, this may indicate that photoactivation ofDNA crosslinking is not stimulated efficiently. Other successfulapplication of PDT is, for example, cardiac ablasion therapy. e.g.,treating cardiac arrhythmias and atrial fibrillation which are believedto be a significant cause of cerebral stroke.

U.S. Pat. No. 6,811,562 describes administering a photoactivatable agentand subjecting cardiac tissue containing the administered agent to laserirradiation having a wavelength from 350 to 700 nm using invasivetechniques, e.g., a fiber optic element.

Yet, another application of PDT is photoangioplasty for arterialdiseases including de novo atherosclerosis and restinosis (Rockson A G,et al., Circulation, 102:591-596 (2000); Hsiang Y N., et al., J.Endovasc. Surg., 2:365-371 (1995)). In human clinical applications,endovascular light (730 nm) is delivered through a cylindrical fiberafter intravenous administration of motexafin lutetium. PDT is also usedfor preventing and treatment of intimal hyperplasia in blood vessels invivo (see, e.g., U.S. Pat. No. 6,609,014).

Age-related macular degeneration (AMD) is a cause of new blindness.Choroidal neovascularization leads to hemorrhage and fibrosis in anumber of ocular diseases. Conventional treatments utilize the argonlaser to occlude the leaking vessel by thermal coagulation. However, thepercentage of patients eligible for this treatment is limited. PDT isused for treating AMD and involves injecting verteporfin followed by theapplication of non-thermal light at 692 nm.

Improvement of clinical appearance of psoriatic plaques andpalmopustular psoriasis using PUVA with hematopotphyrin was firstreported in 1937. Acne, apopecia areata, portwine stains and hairremoval also show promise with PDT treatment.

The choice of therapy usually depends on the location and severity ofthe disorder, the stage of the disease, as well as the patient'sresponse to the treatment.

While some treatments may only seek to manage and alleviate symptoms ofthe disorder, the ultimate goal of any effective therapy is the completeremoval or cure of all disordered cells without damage to the rest ofthe body.

The Photo-spectral Therapy (PST) modality is different (if notcomplementary) to the phototherapy technique often referred toPhoto-thermal Therapy (PTT). The use of plasmonics-enhanced photothermalproperties of metal nanoparticles for photothermal therapy has beenreviewed (Xiaohua Huang & Prashant K. Jain & Ivan H. El-Sayed & MostafaA. El-Sayed, “Plasmonic photothermal therapy (PPTT) using goldnanoparticles”, Lasers in Medical Science, August 2007). The PSTtechnique is based on the radiative processes (fluorescence,phosphotscence, luminescence, Raman, etc) and the PPT method is based onthe radiationless processes (IC, VR and heat conversion) in molecules.

A survey of known treatment methods reveals that these methods tend toface a primary difficulty of differentiating between normal cells andtarget cells when delivering treatment, often due to the production ofsinglet oxygen which is known to be non-selective in its attack ofcells, as well as the need to perform the processes ex vivo, or throughhighly invasive procedures, such as surgical procedures in order toreach tissues more than a few centimeters deep within the subject.Another challenge for non-invasive therapeutic modalities is to havesufficient light energy to excite and photo-activate drug molecules deepinside tissue.

U.S. Pat. No. 5,829,448 describes sequential and simultaneous two photonexcitation of photo-agents using irradiation with low energy photonssuch as infrared or near infrared light (NRI). A single photon andsimultaneous two photon excitation is compared for psoralen derivatives,wherein cells are treated with the photo agent and are irradiated withNRI or UV radiation. The patent suggests that treating with a low energyirradiation is advantageous because it is absorbed and scattered to alesser extent than UV radiation. However, the use of NRI or UV radiationis known to penetrate tissue to only a depth of a few centimeters. Thusany treatment deep within the subject would necessarily require the useof ex vivo methods or highly invasive techniques to allow theirradiation source to reach the tissue of interest. Also, this patentdoes not describe initiation energy sources emitting energy other thanUV, visible, and near infrared energy; energy upgrading other thanwithin the range corresponding to UV and IR light, and downgrading fromhigh to low energy.

Chen et al, J. Nanosci. and Nanotech., 6:1159-1166 (2006); Kim et al.,JACS, 129:2669-2675 (2007); U.S. 2002/0127224; and U.S. Pat. No.4,979,935 each describe methods for treatment using various types ofenergy activation of agents within a subject. However, each suffers fromthe drawback that the treatment is dependent on the production ofsinglet oxygen to produce the desired effect on the tissue beingtreated, and is thus largely indiscriminate in affecting both healthycells and the diseased tissue desired to be treated.

U.S. Pat. No. 6,908,591 discloses methods for sterilizing tissue withirradiation to reduce the level of one or more active biologicalcontaminants or pathogens, such as viruses, bacteria, yeasts, molds,fungi, spores, prions or similar agents responsible, alone or incombination, for transmissible spongiform encephalopathies and/or singleor multicellular parasites, such that the tissue may subsequently beused in transplantation to replace diseased and/or otherwise defectivetissue in an animal. The method may include the use of a sensitizer suchas psoralen, a psoralen-derivative or other photosensitizer in order toimprove the effectiveness of the irradiation or to reduce the exposurenecessary to sterilize the tissue. However, the method is not suitablefor treating a patient and does not teach any mechanisms for stimulatingthe photosensitizers, indirectly.

U.S. Pat. No. 5,957,960 discloses a two-photon excitation device foradministering a photodynamic therapy to a treatment site within apatient's body using light having an infrared or near infrared waveband.However, the reference fails to disclose any mechanism ofphotoactivation using energy modulation agent that converts theinitiation energy to an energy that activates the activatablepharmaceutical agent and also use of other energy wavebands, e.g.,X-rays, gamma-rays, electron beam, microwaves or radio waves.

U.S. Pat. No. 6,235,508 discloses antiviral applications for psoralensand other photoactivatable molecules. It teaches a method forinactivating viral and bacterial contaminants from a biologicalsolution. The method includes mixing blood with a photosensitizer and ablocking agent and irradiating the mixture to stimulate thephotosensitizer, inactivating substantially all of the contaminants inthe blood, without destroying the red blood cells. The blocking agentprevents or reduces deleterious side reactions of the photosensitizer,which would occur if not in the presence of the blocking agent. The modeof action of the blocking agent is not predominantly in the quenching ofany reactive oxygen species, according to the reference.

Also, U.S. Pat. No. 6,235,508 suggests that halogenated photosensitizersand blocking agents might be suitable for replacing 8-methoxypsoralen(8-MOP) in photopheresis and in treatment of certain proliferativecancers, especially solid localized tumors accessible via a fiber opticlight device or superficial skin cancers. However, the reference failsto address any specific molecules for use in treating lymphomas or anyother cancer. Instead, the reference suggests a process of photopheresisfor antiviral treatments of raw blood and plasma.

U.S. Pat. No. 6,235,508 teaches away from 8-MOP and4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) and many otherphotoactivatable molecules, which are taught to have certaindisadvantages. Fluorescing photosensitizers are said to be preferred,but the reference does not teach how to select a system of fluorescentstimulation or photoactivation using fluorescent photosensitizers.Instead, the fluorescing photosensitizer is limited to the intercalatorthat is binding to the DNA. The reference suggests that fluorescenceindicates that such an intercalator is less likely to stimulate oxygenradicals.

U.S. published application 2002/0127224 discloses a method for aphotodynamic therapy comprising administering light-emittingnanoparticles and a photoactivatable agent, which may be activated bythe light re-emitted from the nanoparticles via a two-photon activationevent. An initiation energy source is usually a light emitting diode,laser, incandescent lamp, or halogen light, which emits light having awavelength ranging from 350 to 1100 nm. The initiation energy isabsorbed by the nanoparticles. The nanoparticles, in turn, re-emit lighthaving a wavelength from 500 to 1100 nm, preferably, UV-A light, whereinthe re-emitted energy activates the photoactivatable agent. Kim et al.,(JACS, 129:2669-75, Feb. 9, 2007) discloses indirect excitation of aphotosensitizing unit (energy acceptor) through fluorescence resonanceenergy transfer (FRET) from the two-photon absorbing dye unit (energydonor) within an energy range corresponding to 300-850 nm. Thesereferences do not describe initiation energy sources emitting energyother than UV, visible, and near infrared energy; energy upgrading otherthan within the range corresponding to wavelength of 350-1100 nm, anddowngrading from high to low energy.

These references fail to disclose any mechanism of photoactivation of anphotoactivatable molecules other than by direct photoactivation by UV,visible, and near infrared energy.

Psoralens and Related Compound

U.S. Pat. No. 6,235,508 further teaches that psoralens are naturallyoccurring compounds which have been used therapeutically for millenniain Asia and Africa. The action of psoralens and light has been used totreat vitiligo and psoriasis (PUVA therapy; Psoralen Ultra Violet A).Psoralen is capable of binding to nucleic acid double helices byintercalation between base pairs; adenine, guanine, cytosine and thymine(DNA) or uracil (RNA). Upon sequential absorption of two UV-A photons,psoralen in its excited state reacts with a thymine or uracil doublebond and covalently attaches to both strands of a nucleic acid helix.The crosslinking reaction appears to be specific for a thymine (DNA) ora uracil (RNA) base. Binding proceeds only if psoralen is intercalatedin a site containing thymine or uracil, but an initial photoadduct mustabsorb a second UVA photon to react with a second thymine or uracil onthe opposing strand of the double helix in order to crosslink each ofthe two strands of the double helix, as shown below. This is asequential absorption of two single photons as shown, as opposed tosimultaneous absorption of two or more photons.

In addition, the reference teaches that 8-MOP is unsuitable for use asan antiviral, because it damages both cells and viruses. Lethal damageto a cell or virus occurs when the psoralen is intercalated into anucleic acid duplex in sites containing two thymines (or uracils) onopposing strands but only when it sequentially absorbs 2 UVA photons andthymines (or uracils) are present. U.S. Pat. No. 4,748,120 of Wiesehanis an example of the use of certain substituted psoralens by aphotochemical decontamination process for the treatment of blood orblood products.

Additives, such as antioxidants are sometimes used with psoralens, suchas 8-MOP, AMT and I-IMT, to scavenge singlet oxygen and other highlyreactive oxygen species formed during photoactivation of the psoralens.It is well known that UV activation creates such reactive oxygenspecies, which are capable of seriously damaging otherwise healthycells. Much of the viral deactivation may be the result of thesereactive oxygen species rather than any effect of photoactivation ofpsoralens. Regardless, it is believed that no auto vaccine effect hasbeen observed.

The best known photoactivatable compounds are derivatives of psoralen orcoumarin, which are nucleic acid intercalators. The use of psoralen andcourmarin photosensitizers can give rise to alternative chemicalpathways for dissipation of the excited state that are either notbeneficial to the goal of viral inactivation, or that are actuallydetrimental to the process. For psoralens and coumarins, this chemicalpathway is likely to lead to the formation of a variety of ring-openedspecies, such as shown below for coumarin:

Research in this field over-simplifies mechanisms involved in thephotoactivating mechanism and formation of highly reactive oxygenspecies, such as singlet oxygen. Both may lead to inactivating damage oftumor cells, viruses and healthy cells. However, neither, alone orcombined, lead to an auto vaccine effect. This requires an activation ofthe body's own immune system to identify a malignant cell or virus asthreat and to create an immune response capable of lasting cytotoxiceffects directed to that threat. It is believed, without being limitingin any way, that photoactivation and the resulting apoptosis ofmalignant cells that occurs in extracorporeal photophoresis causes theactivation of an immune response with cytotoxic effects on untreatedmalignant cells. While the complexity of the immune response andcytotoxic effects is fully appreciated by researchers, a therapy thatharnesses the system to successfully stimulate an auto vaccine effectagainst a targeted, malignant cell has been elusive, except forextracorporeal photopheresis for treating lymphoma.

Midden (W. R. Midden, Psoralen DNA photobiology, Vol II (ed. F. P.Gaspalloco) CRC press, pp. 1. (1988) has presented evidence thatpsoralens photoreact with unsaturated lipids and photoreact withmolecular oxygen to produce active oxygen species such as superoxide andsinglet oxygen that cause lethal damage to membranes. U.S. Pat. No.6,235,508 teaches that 8-MOP and AMT are unacceptable photosensitizers,because each indiscriminately damages both cells and viruses. Studies ofthe effects of cationic side chains on furocoumarins as photosensitizersare reviewed in Psoralen DNA Photobiology, Vol. I, ed. F. Gaspano, CRCPress, Inc., Boca Raton, Fla., Chapter 2. U.S. Pat. No. 6,235,508 gleansthe following from this review: most of the amino compounds had a muchlower ability to both bind and form crosslinks to DNA compared to 8-MOP,suggesting that the primary amino functionality is the preferred ionicspecies for both photobinding and crosslinking.

U.S. Pat. No. 5,216,176 of Heindel discloses a large number of psoralensand coumarins that have some effectiveness as photoactivated inhibitorsof epidermal growth factor. Halogens and amines are included among thevast functionalities that could be included in the psoralen/coumarinbackbone. This reference is incorporated herein by reference.

U.S. Pat. No. 5,984,887 discloses using extracorporeal photopheresiswith 8-MOP to treat blood infected with CMV. The treated cells as wellas killed and/or attenuated virus, peptides, native subunits of thevirus itself (which are released upon cell break-up and/or shed into theblood) and/or pathogenic noninfectious viruses are then used to generatean immune response against the virus, which was not present prior to thetreatment.

Problems with PDT

It is well recognized that a major problem associated with the existingmethods of diagnosis and treatment of cell proliferation disorders is indifferentiation of normal cells from target cells. Radiation therapyworks by irradiating cells with high levels of high energy radiationsuch as high energy photon, electron, or proton. These high energy beamsionize the atoms which make up a DNA chain, which in turn leads to celldeath. Unlike surgery, radiation therapy does not require placingpatients under anesthesia and has the ability to treat disorders deepinside the body with minimal invasion of the body. However, the highdoses of radiation needed for such therapies damages healthy cells justas effectively as it does diseased cells. Thus, similar to surgery,differentiation between healthy and diseased cells in radiation therapyis only by way of location. There is no intrinsic means for a radiationbeam to differentiate between a healthy cell from a diseased celleither. Another problem encountered in PDT therapy is the inability totreat target areas that are more than a few centimeters beneath thesurface of the skin without significant invasive techniques. Anotherchallenge for non-invasive therapeutic modalities is to have sufficientlight energy to excite and photo-activate drug molecules deep insidetissue.

Therefore, there still exists a need for better and more effectivetreatments that can more precisely target the diseased cells withoutcausing substantial side-effects or collateral damages to healthytissues, and which are capable of treating disorders by non-invasive orminimum invasive techniques.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodfor modifying a target structure which mediates or is associated with abiological activity in a subject that permits treatment of a subject inany area of the body while being non-invasive and having highselectivity for targeted cells relative to healthy cells using energyupconversion.

A further object of the present invention is to provide a method formodifying a target structure which mediates or is associated with abiological activity which can use any suitable energy source as aninitiation energy source to induce a predetermined change in a targetstructure in a subject in situ to treat a condition, disorder ordisease.

A further object of the present invention is to provide a method formodifying a target structure which mediates or is associated with abiological activity using a modulation agent which converts aninitiation energy into an energy that causes nanoparticles placed in thevicinity of a target structure to emit light that induces apredetermined change in the target structure.

A further object of the present invention is to provide a method formodifying a target structure which mediates or is associated with abiological activity using a modulation agent which converts the energyemitted by the nanoparticles placed in the vicinity of a, targetstructure so that the energy reemitted by the energy modulation agentinduces a predetermined change in the target structure.

These and other objects of the present invention, which will become moreapparent in conjunction with the following detailed description of thepreferred embodiments, either alone or in combinations thereof, havebeen satisfied by the discovery of a method for modifying a targetstructure which mediates or is associated with a biological activity,comprising:

placing a nanoparticle in a vicinity of a target structure in a subjectin need of treatment, wherein the nanoparticle is configured, uponexposure to a first wavelength λ₁, to generate a second wavelength λ₂ ofradiation having a higher energy than the first wavelength λ₁, wherein

-   -   the nanoparticle comprises a metallic shell on at least a        fraction of a surface of the nanoparticle,    -   a radial dimension of the metallic shell is set to a value so        that a surface plasmon resonance in the metallic shell resonates        at a frequency which provides spectral overlap with at least one        of the first wavelength λ1 and the second wavelength λ2, and    -   the nanoparticle is configured to emit light in the vicinity of        or into the target structure upon interaction with an initiation        energy having an energy in the range of λ1; and

applying the initiation energy including said first wavelength λ1 froman initiation energy source to the subject, wherein the emitted lightincluding said second wavelength λ2 directly or indirectly contacts thetarget structure and induces a predetermined change in said targetstructure in situ,

wherein said predetermined change modifies the target structure andmodulates the biological activity of the target structure.

Yet a further object of the invention is further administer at least oneenergy modulation agent to said subject which converts said initiationenergy into an energy that effects a predetermined change in said targetstructure.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease which can use any suitableenergy source as the initiation energy source to activate theactivatable pharmaceutical agent and thereby cause a predeterminedchange in a target structure to treat a condition, disorder or disease.

A further object of the present invention is to provide a method fortreatment of a condition, disorder or disease using an energy cascade toactivate an activatable pharmaceutical agent that then treats cellssuffering from a condition, disorder or disease.

A further object of the present invention is to provide a method forgenerating an autovaccine effect in a subject, which can be in vivo thusavoiding the need for ex vivo treatment of subject tissues or cells, orcan be ex vivo.

A further object of the present invention is to provide a method forgenerating an autovaccine effect in a subject, which can be in vivo thusavoiding the need for ex vivo treatment of subject tissues or cells, orcan be ex vivo.

A further object of the present invention is to provide a method for ormodifying a target structure which mediates or is associated with abiological activity, comprising:

-   -   a. modifying one or more cells to incorporate a photon emitting        modification or substance;    -   b. inserting the modified cells at a targeted site of the        subject;    -   c. placing in a vicinity of a target structure in a subject in        need of treatment a nanoparticle, the nanoparticle is        configured, upon exposure to the photons emitted from the        modified cells having a first wavelength λ₁, to generate a        second wavelength λ₂ of radiation having a higher energy than        the first wavelength λ₁, wherein        -   the nanoparticle includes a metallic shell on at least a            fraction of a surface of the nanoparticle,        -   a radial dimension of the metallic shell is set to a value            so that a surface plasmon resonance in the metallic shell            resonates at a frequency which provides spectral overlap            with at least one of the first wavelength λ₁ and the second            wavelength λ₂, and        -   the nanoparticle is configured to emit energy upon            interaction with an initiation energy having an energy in            the range of λ₁;    -   d. administering (i) at least one activatable pharmaceutical        agent capable of being activated directly or indirectly by the        energy emitted by the nanoparticle to cause a predetermined        change to the target structure in situ, and (ii) optionally, at        least one energy modulation agent;        -   thus causing the predetermined change to the target            structure to occur, wherein said predetermined change            modifies the target structure and modulates the biological            activity of the target structure.

A still further object of the present invention is to provide a kit, asystem, and a pharmaceutical composition for use in the presentinvention methods.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 provides an exemplary electromagnetic spectrum in meters.

FIG. 2 is a graphical representation of an embodiment of the energymodulation agent-photo activator (PA) system of the present invention.

FIG. 3 is a graphical representation of the “therapeutic window” intissue and absorption spectra of biological components.

FIGS. 4A and 4B are graphical representations of plasmonicnanostructures and their theoretical electromagnetic enhancement atdifferent excitation wavelengths.

FIG. 5 is an energy diagram of an infrared phosphor system.

FIG. 6 is a schematic energy level diagram showing upconversionexcitation and visible emissions schemes for Er³⁺, Tm³⁺ and or Yb³⁺ions.

FIG. 7 is an energy diagram showing energy states for a four-photonupconversion process in Y₂O₃ nanocrystals.

FIGS. 8A-A-8A-G are schematic illustrations of various upconverterstructures of the invention.

FIG. 8A-1 is an UV-visible absorption spectra of cubic Y₂O₃ andgold-coated Y₂O₃ dispersed using 10 mM tri-arginine.

FIG. 8B is a schematic illustration of plasmon resonance as a functionof shell thickness.

FIG. 8C is a schematic illustration of a process for forming and aresultant Ln-doped Y₂O₃ core with a Au shell.

FIG. 8D is a schematic illustration of a process for forming and aresultant Ln-doped Y₂O₃ core with a NaYF₄ shell.

FIG. 9 is a schematic illustration of a particular nanometer sizedupconverter structure of the invention.

FIGS. 9-1-A and 9-1-B are a micrograph showing ˜15 nm cubic Y₂O₃dielectric particles generated through the combustion method(magnifications A and B).

FIG. 9-2 is a micrograph showing NaYF₄ dielectric particles in the sizerange of ˜70-200 nm range.

FIG. 9-3 is a micrograph showing NaYF₄ dielectric particles with twosize distributions of ˜50 nm and ˜150 nm.

FIG. 9-4 is a micrograph showing YbF₃ dielectric particles of a size of35 nm+/−5 nm.

FIG. 9-5 is an optical emission spectrum from YbF₃; Tm (2%) dielectricparticles, excited at 980 nm.

FIGS. 9-6, 9-7, 9-8, and 9-9 are micrographs showing NaYbF₄ dielectricparticles in the ˜20-150 nm size range.

FIGS. 10A-A-10A-G are schematic illustrations of other variousupconverter structures of the invention.

FIGS. 10B-A-10B-G are schematic illustrations of other variousupconverter structures of the invention.

FIGS. 10C-A-10C-J are schematic illustrations of plasmonics-activeupconverter structures of the invention.

FIGS. 10D-A-10D-G are schematic illustrations of photo-active moleculeslinked to plasmonics-active upconverter structures of the invention.

FIG. 10E is a TEM micrograph of uncoated Y₂O₃ nanoparticles.

FIG. 10F is a TEM micrograph of gold coated Y₂O₃ nanoparticles of theinvention.

FIG. 10G is X-ray diffraction data from gold coated Y₂O₃ nanoparticlesof the invention.

FIG. 10H is a TEM micrograph of 15-nm gold nanoparticles preparedaccording to one embodiment of the present invention using the citratereduction technique.

FIG. 10I is a TEM micrograph of 30-nm gold nanoparticles preparedaccording to one embodiment of the present invention using the citratereduction technique.

FIG. 10J is a TEM micrograph of 60-nm gold nanoparticles preparedaccording to one embodiment of the present invention using the citratereduction technique.

FIG. 10K is a TEM micrograph of 30-nm gold nanoparticles preparedaccording to one embodiment of the present invention using the hydrazinemonohydrate reduction technique.

FIG. 10L is a TEM micrograph of silver nanoparticles formed by and usedin the present invention.

FIG. 10M is a TEM micrograph of Au coated with Ag nanoparticles formedby and used in the present invention.

FIG. 10N is a TEM micrograph of Au/Ag/Au/Ag multi-shell nanoparticlesformed by and used in the present invention.

FIGS. 11A-11C are schematic illustrations of other various upconverterstructures of the invention where a recipient molecule is bound to themetal nanoparticles via a linker that can be dissociated by a photonradiation.

FIGS. 12A-A-12A-G are schematic illustrations of other variousupconverter structures of the invention where the dielectric core hasappended thereon or attached by linkages a bioreceptor molecule.

FIGS. 12B-A-12B-F are schematic illustrations of still other variousupconverter structures of the invention where the dielectric core hasappended thereon or attached by linkages a bioreceptor molecule.

FIG. 12B-1 is a depiction of the enhancement of emission as a functionof wavelength for a configuration similar to that in FIG. 12B-F.

FIG. 123B-2 is a depiction of the enhancement of emission as a functionof wavelength for a configuration where the molecule is located inside ametallic shell.

FIG. 12B-3 is a depiction of the excitation enhancement as a function ofwavelength for a configuration similar to that in FIG. 12A-F.

FIG. 1.2B-4 is a depiction of the dependence of emission enhancement onwavelength for the structure and excitation shown in FIG. 12B-3.

FIG. 12B-5 is a depiction of the data of FIG. 12B-4 simplified to showthe total enhancement verses the inside diameter of the metal shell.

FIG. 13A, B, C is a graphical representation of an embodiment of a PEPSTenergy modulation agent-PA system with detachable bond.

FIG. 14 is a graphical representation of an embodiment of PEPST probesfor dual plasmonic excitation.

FIGS. 15A, B, C, and D are graphical representations of an embodiment ofa use of encapsulated photoactive agents.

FIGS. 16A and 16B are simplified graphical representations of the use ofthe present invention principle of non-invasive PEPST modality.

FIGS. 17A and 17B show various schematic embodiments of basic EIPprobes.

FIGS. 18A and 18B are graphical representations of various embodimentsof basic EPEP probes.

FIGS. 19A-19C are graphical representations of various embodiments ofbasic EPEP probes.

FIGS. 20A and 20B are graphical representations of various embodimentsof EPEP probes having NPs, NWs and NRs.

FIGS. 21A and 21B are graphical representations of various embodimentsof EPEP probes having NPs, NWs, NRs and bioreceptors.

FIG. 22 is a graphical representation of an embodiment of EPEP probeshaving NPs and multiple NWs.

FIG. 23 is a depiction of both down conversion and up conversionemission from a thulium doped nanoparticle (NaYbF₄; 3% Tm).

FIG. 24 is a micrograph of a representative 35 nm PEI Coated YbF₃; Tm(2%) particle.

FIG. 25A is a schematic depicting a system according to anotherembodiment of the invention in which the initiation energy source isdirected to a medium having energy modulation agents disbursed withinthe medium;

FIG. 25B is a schematic depicting a system according to anotherembodiment of the invention in which the initiation energy source isdirected to a container enclosing a medium having energy modulationagents disbursed within the medium;

FIG. 25C is a schematic depicting a system according to anotherembodiment of the invention in which the initiation energy source isdirected to a container enclosing a medium having energy modulationagents segregated within the medium; and

FIG. 25D is a schematic depicting a system according to anotherembodiment of the invention in which the initiation energy source isdirected to a container enclosing a medium having energy modulationagents segregated within the medium in a fluidized bed configuration.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to methods and systems for producingelectromagnetic radiation having desirable frequency windows (at leastone frequency within a desirable frequency range) from otherelectromagnetic radiation having lower or higher frequency ranges usingup converting transitional media or down converting transitional mediaas the case may apply. In various embodiments of the invention, theproduced electromagnetic radiation is then be used to activate an agentin a medium where the up converting transitional media or downconverting transitional media are disposed. In various embodiments, theapplied energy is considered to be up converted, as the photon energycarried by radiation 1 has an energy level equal to hv₁ (the product ofPlanck constant and frequency 1) is converted to a higher energy hv₂,where hv₁ is less than hv₂. In various embodiments, the applied energyis considered to be down converted, as energy at hv1, is converted to alower energy hv₂, where hv₁ is greater than hv₂.

In various embodiments of the invention, there are provided systems andmethods for broad band up conversion from the microwave and RF regime toelectromagnetic radiation of higher photonic energy in the UV, VIS, andIR regime.

The invention can encompasses a variety of applications where the up anddown conversions are conducted inside biological media (or) inside humanand animal bodies; in chemical reactors and/or in semiconductors andsolar cells to name but a few.

The present invention in biological media sets forth a novel method formodifying a target structure in a subject which mediates or isassociated with a biological activity that is effective, specific, andhas few side-effects. Those cells suffering from a condition, disorderor disease are referred to herein as the target cells.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

In one embodiment, the present invention provides a method for modifyinga target structure which mediates or is associated with a biologicalactivity, comprising:

placing a nanoparticle in a vicinity of a target structure in a subjectin need of treatment, wherein the nanoparticle is configured, uponexposure to a first wavelength λ₁, to generate a second wavelength 2 ofradiation having a higher energy than the first wavelength λ₁, wherein

-   -   the nanoparticle comprises a metallic structure is deposited in        relation to the nanoparticle,    -   a radial dimension of the metallic shell is set to a value so        that a surface plasmon resonance in the metallic shell resonates        at a frequency which provides spectral overlap with at least one        of the first wavelength λ1 and the second wavelength λ2, and    -   the nanoparticle is configured to emit light in the vicinity of        or into the target structure upon interaction with an initiation        energy having an energy in the range of λ1; and

applying the initiation energy including said first wavelength λ1 froman initiation energy source to the subject, wherein the emitted lightincluding said second wavelength λ2 directly or indirectly contacts thetarget structure and induces a predetermined change in said targetstructure in situ,

wherein said predetermined change modifies the target structure andmodulates the biological activity of the target structure.

In one embodiment, the initiation energy produces plasmonics and/orexciton coupling enhanced light generation that is capable of inducing apredetermined change in the target structure with or without an energymodulation agent and/or a photoactive agent.

In various embodiments, the applied energy is considered to be upconverted, as the photon energy carried by radiation 1 has an energylevel equal to hv₁ (the product of Planck constant and frequency 1) isconverted to a higher energy hv₂, where hv₁ is less than hv₂. In variousembodiments, the applied energy is considered to be down converted, asenergy at hv₁, is converted to a lower energy hv₂, where hv₁ is greaterthan hv₂.

In various embodiments of the invention, there are provided systems andmethods for broad band up conversion from the microwave and RF regime toelectromagnetic radiation of higher photonic energy in the UV, VIS, andIR regime. The invention can encompasses a variety of applications wherethe up and down conversions are conducted inside biological media (or)inside human and animal bodies.

Among various materials, luminescent nanoparticles have attractedincreasing technological and industrial interest. In the context of theinvention, nanoparticle refers to a particle having a size less than onemicron. While the description of the invention describes specificexamples using nanoparticles, the invention in many embodiments is notlimited to particles having a size less than one micron. However, inmany of the embodiments, the size range of having a size less than onemicron, and especially less than 100 nm produces properties of specialinterest such as for example emission lifetime luminescence quenching,luminescent quantum efficiency, and concentration quenching and such asfor example diffusion, penetration, and dispersion into mediums wherelarger size particles would not migrate.

As noted above, an object of the present invention is to modify a targetstructure which mediates or is associated with a biological activity,and in one preferred embodiment to treat a condition, disorder ordisease, in a subject using photobiomodulation.

In one preferred embodiment, the initiation energy source is appliedindirectly via an energy modulation agent, preferably in proximity tothe target cells. The present invention further provides methods for thetreatment of a condition, disorder or disease, in which at least oneenergy modulation agent converts the initiation energy into an energy inthe range of the first wavelength λ₁ that cause the nanoparticle to emitlight in the range of the second wavelength λ₂ that is capable ofinducing the predetermined change in said target structure. In a otherembodiment, the at least one energy modulation agent converts theinitiation energy into an energy in the range of the first wavelength λ₁that cause the nanoparticle to emit energy in the range of said secondwavelength λ₂ that is capable of inducing the predetermined change inthe target structure. In one preferred embodiment, the energy modulationagent is specifically located around, on, or in said target structure.In yet another embodiment, the energy modulation agent transforms theinitiation electromagnetic energy into a photonic or anotherelectromagnetic energy the range of the first wavelength λ₁ that causethe nanoparticle to emit light the range of said second wavelength λ₂that is capable of of inducing the predetermined change in said targetstructure. In one embodiment, the energy modulation agent is capable ofdownconverting the initiation energy. In another embodiment, the energymodulation agent is capable of upconverting the initiation energy.

As noted above, an object of the present invention is to modify a targetstructure which mediates or is associated with a biological activity,and in a preferred embodiment to treat a condition, disorder or disease,in a subject using photobiomodulation. Exemplary conditions, disordersor diseases may include, but are not limited to, cancer (e.g., prostate,breast, lung, and colon), autoimmune diseases, soft and bone tissueinjury, chronic pain, wound healing, nerve regeneration, viral andbacterial infections, fat deposits (liposuction), varicose veins,enlarged prostate, retinal injuries and other ocular diseases,Parkinson's disease, and behavioral, perceptional and cognitivedisorders. Exemplary conditions also may include nerve (brain) imagingand stimulation, a direct control of brain cell activity with light,control of cell death (apoptosis), and alteration of cell growth anddivision.

Accordingly, in one embodiment, the present invention provides methodsthat are capable of overcoming the shortcomings of the existing methods.In general, a method in accordance with the present invention utilizesan initiation energy from at least one source applied to a targetstructure in a subject in need of treatment, wherein the initiationenergy indirectly contacts the target structure and induces apredetermined change in said target structure in situ, thus modifying atarget structure which mediates or is associated with a biologicalactivity, preferably treating a condition, disorder or disease. Theinitiation energy can preferably penetrate completely through thesubject and can be applied from a single source or more than one source.Exemplary initiation energy may be UV radiation, visible light, infraredradiation (IR), x-rays, gamma rays, an electron beam, microwaves orradio waves.

In one embodiment, a plasmonics-active agent (e.g., a nanoparticle)upconverts the applied initiation energy, such that the upconvertedinitiation energy is capable of inducing the predetermined change insaid target structure. “Energy upconversion” means that upon exposure toa first wavelength λ₁, an agent (e.g., energy modulation agent orplasmonics active agent or both) generates a second wavelength λ₂ ofradiation having a higher energy than the first wavelength λ₁. In adifferent embodiment, an energy modulation agent upconverts the appliedinitiation energy, such that the upconverted initiation energy isabsorbed, intensified or modified by at least one plasmonics-activeagent into an energy that effects the predetermined change in saidtarget structure. In another embodiment, the initiation energy isabsorbed, intensified or modified by at least one plasmonics activeagent into energy capable to be upconverted by an energy modulationagent (e.g., a nanoparticle) into an energy that is capable of inducingthe predetermined change in said target structure. In yet anotherpreferred embodiment, a method in accordance with the present inventionutilizes the principle of energy transfer to and among molecular agentsto control delivery and activation of cellular changes by irradiationsuch that delivery of the desired effect is more intensified, precise,and effective than the conventional techniques.

Further, the energy modulation agent can transform a photonic initiationenergy into a photonic energy that effects a predetermined change insaid target structure. In one preferred embodiment, the energymodulation agent downconverts the wavelength of the photonic initiationenergy. In another preferred embodiment, the energy modulation agent canupconvert the wavelength of the photonic initiation energy. In adifferent embodiment the modulation agent is one or more membersselected from a biocompatible fluorescing metal nanoparticle,fluorescing metal oxide nanoparticle, fluorescing dye molecule, goldnanoparticle, silver nanoparticle, gold-coated silver nanoparticle, awater soluble quantum dot encapsulated by polyamidoamine dendrimers, aluciferase, a biocompatible phosphorescent molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelateexhibiting intense luminescence.

Another object of the present invention is to treat a condition,disorder or disease in a subject using an activatable pharmaceuticalagent. Exemplary conditions, disorders or diseases may include, but arenot limited to, cancer, autoimmune diseases, cardiac ablasion (e.g.,cardiac arrhythmia and atrial fibrillation), photoangioplasticconditions (e.g., de novo atherosclerosis, restinosis), intimalhyperplasia, arteriovenous fistula, macular degeneration, psoriasis,acne, hopecia areata, portwine spots, hair removal, rheumatoid andinflammatory arthritis, joint conditions, lymph node conditions, andcognitive and behavioral conditions.

Accordingly, in one embodiment, the present invention provides methodsutilizing the principle of energy transfer to and among molecular agentsto control delivery and activation of pharmaceutically active agentssuch that delivery of the desired pharmacological effect is morefocused, precise, and effective than the conventional techniques.

In yet another preferred embodiment, the initiation energy source isapplied directly or indirectly to the activatable pharmaceutical agentand/or a plasmonics-active agent (e.g., nanoparticles), preferably inproximity to the target cells.

Within the context of the present invention, the phrase “appliedindirectly” (or variants of this phrase, such as “applying indirectly”,“indirectly applies”, “indirectly applied”, “indirectly applying”,etc.), when referring to the application of the initiation energy, meansthe penetration by the initiation energy into the subject beneath thesurface of the subject and to the modulation agent and/or activatablepharmaceutical agent within a subject. In one embodiment, the initiationenergy source cannot be within line-of-sight of the energy modulationagent and/or the activatable pharmaceutical agent. By “cannot be withinline-of-sight” is meant that if a hypothetical observer were located atthe location of the energy modulation agent or the activatablepharmaceutical agent, that observer would be unable to see the source ofthe initiation energy.

Although not intending to be bound by any particular theory or beotherwise limited in any way, the following theoretical discussion ofscientific principles and definitions are provided to help the readergain an understanding and appreciation of the present invention.

As used herein, the term “subject” is not intended to be limited tohumans, but may also include animals, plants, or any suitable biologicalorganism.

As used herein, the phrase “a disease or condition” refers to acondition, disorder or disease that may include, but are not limited to,cancer, soft and bone tissue injury, chronic pain, wound healing, nerveregeneration, viral and bacterial infections, fat deposits(liposuction), varicose veins, enlarged prostate, retinal injuries andother ocular diseases, Parkinson's disease, and behavioral, perceptionaland cognitive disorders. Exemplary conditions also may include nerve(brain) imaging and stimulation, a direct control of brain cell activitywith light, control of cell death (apoptosis), and alteration of cellgrowth and division. Yet other exemplary a condition, disorder ordisease may include, but are not limited to, cardiac ablasion (e.g.,cardiac arrhythmia and atrial fibrillation), photoangioplasticconditions (e.g., de novo atherosclerosis, restinosis), intimalhyperplasia, arteriovenous fistula, macular degeneration, psoriasis,acne, hopecia areata, portwine spots, hair removal, rheumatoid andinflammatory arthritis, joint conditions, and lymph node conditions.

As used herein, the term “target structure” refers to an eukaryoticcell, prokaryotic cell, a subcellular structure, such as a cellmembrane, a nuclear membrane, cell nucleus, nucleic acid, mitochondria,ribosome, or other cellular organelle or component, an extracellularstructure, virus or prion, and combinations thereof.

The nature of the predetermined cellular change will depend on thedesired pharmaceutical outcome. Exemplary cellular changes may include,but are not limited to, apoptosis, necrosis, up-regulation of certaingenes, down-regulation of certain genes, secretion of cytokines,alteration of cytokine receptor responses, regulation of cytochrome coxidase and flavoproteins, activation of mitochondria, stimulationantioxidant protective pathway, modulation of cell growth and division,alteration of firing pattern of nerves, alteration of redox properties,generation of reactive oxygen species, modulation of the activity,quantity, or number of intracellular components in a cell, modulation ofthe activity, quantity, or number of extracellular components producedby, excreted by, or associated with a cell, or a combination thereof.Predetermined cellular changes may or may not result in destruction orinactivation of the target structure.

As used herein, an “energy modulation agent” refers to an agent that iscapable of receiving an energy input from a source and then re-emittinga different energy to a receiving target. Energy transfer amongmolecules may occur in a number of ways. The form of energy may beelectronic, thermal, electromagnetic, kinetic, or chemical in nature.Energy may be transferred from one molecule to another (intermoleculartransfer) or from one part of a molecule to another part of the samemolecule (intramolecular transfer). For example, a modulation agent mayreceive electromagnetic energy and re-emit the energy in the form ofthermal energy. In preferred embodiments, the energy modulation agentreceives higher energy (e.g. x-ray) and re-emits in lower energy (e.g.UV-A). Some modulation agents may have a very short energy retentiontime (on the order of fs, e.g. fluorescent molecules) whereas others mayhave a very long half-life (on the order of minutes to hours, e.g.luminescent or phosphorescent molecules). Suitable energy modulationagents include, but are not limited to, a biocompatible fluorescingmetal nanoparticle, fluorescing dye molecule, gold nanoparticle, a watersoluble quantum dot encapsulated by polyamidoamine dendrimers, aluciferase, a biocompatible phosphorescent molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelatecapable of intense luminescence. Various exemplary uses of these aredescribed below in preferred embodiments.

The modulation agents may further be coupled to a carrier for cellulartargeting purposes. For example, a biocompatible molecule, such as afluorescing metal nanoparticle or fluorescing dye molecule that emits inthe UV-A band, may be selected as the energy modulation agent.

The energy modulation agent may be preferably directed to the desiredsite (e.g. a tumor) by systemic administration to a subject. Forexample, a UV-A emitting energy modulation agent may be concentrated inthe tumor site by physical insertion or by conjugating the UV-A emittingenergy modulation agent with a tumor specific carrier, such as a lipid,chitin or chitin-derivative, a chelate or other functionalized carrierthat is capable of concentrating the UV-A emitting source in a specifictarget tumor.

Additionally, the energy modulation agent can be used alone or as aseries of two or more energy modulation agents wherein the energymodulation agents provide an energy cascade. Thus, the first energymodulation agent in the cascade will absorb the activation energy,convert it to a different energy which is then absorbed by the secondenergy modulation in the cascade, and so forth until the end of thecascade is reached with the final energy modulation agent in the cascadeemitting the energy necessary to activate the activatable pharmaceuticalagent and/or to cause a photonics-active agent (e.g., a nanoparticle) toupconvert the reimitted energy.

Exemplary energy modulation agents may include, but are not limited to,at least one energy modulation agent selected from the group consistingof a biocompatible fluorescing metal nanoparticle, fluorescing metaloxide nanoparticle, fluorescing dye molecule, gold nanoparticle, silvernanoparticle, gold-coated silver nanoparticle, a water soluble quantumdot encapsulated by polyamidoamine dendrimers, a luciferase, abiocompatible phosphorescent molecule, a combined electromagnetic energyharvester molecule, and a lanthanide chelate exhibiting intenseluminescence.

As used herein, an “activatable pharmaceutical agent” is an agent thatnormally exists in an inactive state in the absence of an activationsignal. When the agent is activated by a matching activation signalunder activating conditions, it is capable of effecting the desiredpharmacological effect on a target cell (i.e. preferably a predeterminedcellular change).

Signals that may be used to activate a corresponding agent may include,but are not limited to, photons of specific wavelengths (e.g. x-rays,UV, IR, NIR or visible light), electromagnetic energy (e.g. radio ormicrowave), thermal energy, acoustic energy, or any combination thereof.

Activation of the agent may be as simple as delivering the signal to theagent or may further premise on a set of activation conditions. Forexample, in the former case, an activatable pharmaceutical agent, suchas a photosensitizer, may be activated by UV-A radiation. Onceactivated, the agent in its active-state may then directly proceed toeffect a cellular change.

Where activation may further premise upon other conditions, meredelivery of the activation signal may not be sufficient to bring aboutthe desired cellular change. For example, a photoactive compound thatachieves its pharmaceutical effect by binding to certain cellularstructure in its active state may require physical proximity to thetarget cellular structure when the activation signal is delivered. Forsuch activatable agents, delivery of the activation signal undernon-activating conditions will not result in the desired pharmacologiceffect. Some examples of activating conditions may include, but are notlimited to, temperature, pH, location, state of the cell, presence orabsence of co-factors.

Selection of an activatable pharmaceutical agent greatly depends on anumber of factors such as the desired cellular change, the desired formof activation, as well as the physical and biochemical constraints thatmay apply. Exemplary activatable pharmaceutical agents may include, butare not limited to, agents that may be activated by photonic energy,electromagnetic energy, acoustic energy, chemical or enzymaticreactions, thermal energy, or any other suitable activation mechanisms.

When activated, the activatable pharmaceutical agent may effect cellularchanges that include, but are not limited to, apoptosis, redirection ofmetabolic pathways, up-regulation of certain genes, down-regulation ofcertain genes, secretion of cytokines, alteration of cytokine receptorresponses, or combinations thereof.

The mechanisms by which an activatable pharmaceutical agent may achieveits desired effect are not particularly limited. Such mechanisms mayinclude direct action on a predetermined target as well as indirectactions via alterations to the biochemical pathways. A preferred directaction mechanism is by binding the agent to a critical cellularstructure such as nuclear DNA, mRNA, rRNA, ribosome, mitochondrial DNA,or any other functionally important structures. Indirect mechanisms mayinclude releasing metabolites upon activation to interfere with normalmetabolic pathways, releasing chemical signals (e.g. agonists orantagonists) upon activation to alter the targeted cellular response,and other suitable biochemical or metabolic alterations.

The treatment of the present invention can be by the unique methodsdescribed in U.S. application Ser. No. 11/935,655, filed Nov. 6, 2007(incorporated by reference above), or by a modified version of aconventional treatment such as PDT, but using a plasmonics-active agentto enhance the treatment by modifying or enhancing the applied energyor, in the case of using an energy modulation agent, modifying eitherthe applied energy, the emitted energy from the energy modulation agent,or both.

In one preferred embodiment, the activatable pharmaceutical agent iscapable of chemically binding to the DNA or mitochondria at atherapeutically effective amount. In this embodiment, the activatablepharmaceutical agent, preferably a photoactivatable agent, is exposed insitu to an activating energy emitted from an energy modulation agent,which, in turn receives energy from an initiation energy source.

Suitable activatable agents include, but are not limited to, photoactiveagents, sono-active agents, thermo-active agents, andradio/microwave-active agents. An activatable agent may be a smallmolecule; a biological molecule such as a protein, a nucleic acid orlipid; a supramolecular assembly; a nanoparticle; or any other molecularentity having a pharmaceutical activity once activated.

The activatable agent may be derived from a natural or synthetic origin.Any such molecular entity that may be activated by a suitable activationsignal source to effect a predetermined cellular change may beadvantageously employed in the present invention.

Suitable photoactive agents include, but are not limited to: psoralensand psoralen derivatives, pyrene cholesteryloleate, acridine, porphyrin,fluorescein, rhodamine, 16-diazorcortisone, ethidium, transition metalcomplexes of bleomycin, transition metal complexes of deglycobleomycin,organoplatinum complexes, alloxazines such as 7,8-dimethyl-10-ribitylisoalloxazine (riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin),7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide(flavine adenine dinucleotide [FAD]), alloxazine mononucleotide (alsoknown as flavine mononucleotide [FMN] and riboflavine-5-phosphate),vitamin Ks, vitamin L, their metabolites and precursors, andnaphthoquinones, naphthalenes, naphthols and their derivatives havingplanar molecular conformations, porphyrins, dyes such as neutral red,methylene blue, acridine, toluidines, flavine (acriflavinehydrochloride) and phenothiazine derivatives, coumarins, quinolones,quinones, and anthroquinones, aluminum (III) phthalocyaninetetrasulfonate, hematoporphyrin, and phthalocyanine, and compounds whichpreferentially adsorb to nucleic acids with little or no effect onproteins. The term “alloxazine” includes isoalloxazines.

Endogenously-based derivatives include synthetically derived analogs andhomologs of endogenous photoactivated molecules, which may have or lacklower (1 to 5 carbons) alkyl or halogen substituents of thephotosensitizers from which they are derived, and which preserve thefunction and substantial non-toxicity. Endogenous molecules areinherently non-toxic and may not yield toxic photoproducts afterphotoradiation.

Table 1 lists some photoactivatable molecules capable of beingphotoactivated to induce an auto vaccine effect.

TABLE 1 SSET and TTET rate constants for bichromophoric peptidesK_(SSET) (S⁻¹) R_(model)(Å) Compound λ_(ex) (nm) E_(SSET) K_(s) of donor(S⁻¹) K_(SSET) (S⁻¹) (Average) R₀ (Å) R (Å) (Average) E_(TTET) k_(TTET)(S⁻¹) 1B 224 96.3 9.5 × 10° 2.44 × 10⁸  1.87 × 10⁸ 14.7 9 9.5 266 95 1.8× 10⁸ 2.5   5 × 10² 280 94 1.36 × 10⁸  1A 224 80 9.5 × 10° 3.8 × 10⁷3.67 × 10⁷ 14.7 11.8 14.1 266 79 3.6 × 10⁷ 2 3.6 × 10² 280 79 3.6 × 10⁷2B 224 77 9.5 × 10° 3.1 × 10⁷  3.9 × 10⁷ 14.7 11.9 5.5 266 81 3.9 × 10⁷32 9.4 × 10³ 280 83 4.7 × 10⁷ 2A 224 69 9.5 × 10° 2.1 × 10⁷   3 × 10⁷14.7 12.2 8.1 74.3 5.7 × 10⁴ 266 80 3.7 × 10⁷ 280 77 3.2 × 10⁷

Table 2 lists some additional endogenous photoactivatable molecules.

TABLE 2 Biocompatible, endogenous fluorophore emitters. Excitation Max.Emission Max. Endogenous Fluorophores (nm) (nm) Amino acids: Tryptophan280 350 Tyrosine 275 300 Phenylalanine 260 280 Structural Proteins:Collagen 325, 360 400, 405 Elastin 290, 325 340, 400 Enzymes andCoenzymes: flavin adenine dinucleotide 450 535 reduced nicotinamidedinucelotide 290, 351 440, 460 reduced nicotinamide dinucelotidephosphate 336 464 Vitamins: Vitamins A 327 510 Vitamins K 335 480Vitamins D 390 480 Vitamins B₆ compounds: Pyridoxine 332, 340 400Pyridoxamine 335 400 Pyridoxal 330 385 Pyridoxic acid 315 425 Pyrictoxalphosphate  5′-330 400 Vitamin B₁₂ 275 305 Lipids: Phospholipids 436 540,560 Lipofuscin 340-395 540, 430-460 Ceroid 340-395 430-460, 540Porphyrins 400-450 630, 690

FIG. 1 provides an exemplary electromagnetic spectrum in meters (1 nmequals 10⁻⁹ meters).

Although the activatable pharmaceutical agent and the energy modulationagent can be distinct and separate, it will be understood that the twoagents need not be independent and separate entities. In fact, the twoagents may be associated with each other via a number of differentconfigurations. Where the two agents are independent and separatelymovable from each other, they generally interact with each other viadiffusion and chance encounters within a common surrounding medium.Where the activatable pharmaceutical agent and the energy modulationagent are not separate, they may be combined into one single entity.

The initiation energy source can be any energy source capable ofproviding energy at a level sufficient to cause cellular changesdirectly or via a modulation agent which transfer the initiation energyto energy capable of causing the predetermined cellular changes. Also,the initiation energy source can be any energy source capable ofproviding energy at a level sufficient activate the activatable agentdirectly, or to provide the energy to a modulation agent with the inputneeded to emit the activation energy for the activatable agent (indirectactivation). Preferable initiation energy sources include, but are notlimited to, UV-A lamps or fiber optic lines, a light needle, anendoscope, and a linear accelerator that generates x-ray, gamma-ray, orelectron beams. In a preferred embodiment, the initiation energy iscapable of penetrating completely through the subject. Within thecontext of the present invention, the phrase “capable of penetratingcompletely through the subject” is used to refer to energy that canpenetrate to any depth within the subject to activate the activatablepharmaceutical agent. It is not required that the any of the energyapplied actually pass completely through the subject, merely that it becapable of doing so in order to permit penetration to any desired depthto activate the activatable pharmaceutical agent. Exemplary initiationenergy sources that are capable of penetrating completely through thesubject include, but are not limited to, UV light, visible light, IRradiation, x-rays, gamma rays, electron beams, microwaves and radiowaves. In one embodiment, a single or multiple energy sources can beused. The initiation energy can be applied from (i) an initiation energysource that is external to the subject; or (ii) an initiation energysource that is internal to the subject, which is placed or deliveredinternally into the subject and/or the target structure.

An additional embodiment of the present invention is to provide a methodfor treatment of a condition, disease or disorder by the in-situgeneration of energy in a subject in need thereof, where the energygenerated can be used directly to effect a change thereby treating thecondition, disease or disorder, or the energy can be used to activate anactivatable pharmaceutical agent, which upon activation effects a changethereby treating the condition, disease or disorder. The energy can begenerated in-situ by any desired method, including, but not limited to,chemical reaction such as chemiluminescence, or by conversion of anenergy applied to the subject externally, which is converted in-situ toa different energy (of lower or higher energy than that applied),through the use of one or more energy modulation agents.

The phenomenon of ultra weak emission from cellular systems has been atopic of various inquiries since the 1900s. This topic can be tracedback to the early investigations of the Russian biologist GurwitschAlexander G. Gurwitsch more than seventy years ago, who speculated thatultraweak photon emission transmit information in cells [A. G.Gurwitsch, S. S. Grabje, and S. Salkind, “Die Natur des spezifischenErregers der Zellteilung,” Arch. Entwicklungsmech. Org. 100, 11-40,1923].

In the 1970s, this area of research was investigated by a number ofinvestigators. The presence of biological radiation from a variety ofcells was later investigated by several research groups in Europe andJapan using low-noise, sensitive photon-counting detection systems [B.Ruth and F.-A. Popp, “Experimentelle Untersuchungen zur ultraschwachenPhotonenemission biologischer Systeme,” Z. Naturforsch., A: Phys. Sci.31c, 741-745, 1976; T. I. Quickenden and S. S. Que-Hee, “The spectraldistribution of the luminescence emitted during growth of the yeastSaccharomyces cerevisiae and its relationship to mitogenetic radiation,”Photochem. Photobiol. 23, 201-204, 1976; H. Inaba, Y. Shimizu, Y. Tsuji,and A. Yamagishi, “Photon counting spectral analysing system ofextra-weak chemi- and bioluminescence for biochemical applications,”Photochem. Photobiol. 30, 169-175, 1979]. Popp and coworkers suggestedthe evidence of some ‘informational character’ associated with theultra-weak photon emission from biological systems, often referred byPopp as “bio-photons”. Other studies reported ultra-weak photon emissionfrom various species including plant, and animals cells [H. J. Niggli,C. Scaletta, Y. Yan, F.-A. Popp, and L. A. Applegate, “Ultraweak photonemission in assessing bone growth factor efficiency using fibroblasticdifferentiation,” J. Photochem. Photobiol., B, 64, 62-68, 2001;].Results of experiments of U V-irradiated skin fibroblasts indicated thatrepair deficient xeroderma pigmentosum cells show an efficient increaseof ultraweak photon emission in contrast to normal cells, [H. J. Niggli,“Artificial sunlight irradiation induces ultraweak photon emission inhuman skin fibroblasts,” J. Photochem. Photobiol., B 18, 281-285(1993)].

A delayed luminescence emission was also observed in biological systems[F.-A. Popp and Y. Yan, “Delayed luminescence of biological systems interms of coherent states,” Phys. Lett. A 293, 93-97 (2002); A. Scordino,A. Triglia, F. Musumeci, F. Grasso, and Z. Rajfur, “Influence of thepresence of Atrazine in water on in-vivo delayed luminescence ofacetabularium acetabulum,”, J. Photochem. Photobiol., B, 32, 11-17(1996); This delayed luminescence was used in quality control ofvegetable products [A. Triglia, G. La. Malfa, F. Musumeci, C. Leonardi,and A. Scordino, “Delayed luminescence as an indicator of tomato fruitquality,” J. Food. Sci. 63, 512-515 (1998)] or for assessing the qualityor quality changes of biological tissues [Yu Yan, Fritz-Albert Popp *,Sibylle Sigrist, Daniel Schlesinger, Andreas Dolf, Zhongchen Yan, SophieCohen, Amodsen Chotia, “Further analysis of delayed luminescence ofplants”, Journal of Photochemistry and Photobiology B: Biology 78,235-244 (2005)].

It was reported that UV excitation can further enhance the ultra-weakemission and a method for detecting UV-A-laser-induced ultra-weak photonemission was used to evaluate differences between cancer and normalcells. [H. J. Niggli et al, Laser-ultraviolet-A-induced ultraweak photonemission in mammalian cells, Journal of Biomedical Optics 10(2), 024006(2005)].

Accordingly, in one embodiment of the present invention, upon applyingan initiation energy from at least one source to a target structure in asubject in need of treatment, the initiation energy contacts the targetstructure and induces a predetermined change in said target structure insitu,

wherein the predetermined change is the enhancement of energy emissionfrom the target, which then mediates, initiates or enhances a biologicalactivity of other target structures in the subject, or of a second typeof target structure (e.g., a different cell type).

In another embodiment, the initiation energy can itself be energyemitted by at least one cell excited by metabolic processes or someother internal or external trigger, and said applying is conducted viacell-to-cell energy transfer. There are those that maintain that thehealth of the body depends on certain bioelectric vibrations that aresusceptible to chemical or physical toxic factors. Fröhlich notes thatthere are coherent electric vibrations in the frequency range 100 GHz to1 THz, excited in cells by metabolic processes (see Fröhlich H. Coherentelectric vibrations in biological systems and the cancer problem, IEEETransactions on Microwave Theory and Techniques, Vol. MTT-26, No. 8,August, 1978, pp 613-617). This idea is based on observation of theinhibition or stimulation of the growth of yeast and bacteria asfunctions of the applied frequency, showing very stable and repetitiveresonances. If such vibrational states are indeed metabolically excited,then they should be manifested in Raman spectroscopy. Actually, theirexistence has been demonstrated during periods of metabolic activity oflysozyme and E. coli (700 GHz to 5 THz). Emissions have also beenobserved at lower frequencies (150 GHz or less). These vibrations occurin the tissue of higher organisms and they have been hypothesizedexercise some control on cellular growth (see also S. J. Webb et al,Nature, Vol. 218, Apr. 27, 1968, pp. 374-375; and S. J. Webb et al etal, Nature Vol. 222, Jun. 21, 1969, pp. 1199-1200). Cancerization couldresult from a modification of these vibrations by the invasion offoreign molecules, e.g., the presence of free electrons in the conditionbands of proteins. There is some evidence for the presence of doublespectral lines at 1.5 and 6 THz in breast carcinoma, which may be anindication of an interaction between normal cellular vibrations and freeelectrons. In such coherent frequency communication between cells, it isbelieved that the medium through which the communication is transmittedis the water within and around the cells (see Smith, CoherentFrequencies, Consciousness and the Laws of Life, 9^(th) InternationalConference CASYS '09 on Computing Anticipatory Systems, Liege, Belgium,Aug. 3-8, 2009).

Accordingly, in a further embodiment of the present invention, theinitiation energy is an energy capable of triggering an alteredmetabolic activity in one or more cells, preferably in the 100 GHz to 10THz region, and is applied directly to one or more cells, to trigger thecell(s) to undergo altered metabolic activity, and optionally, tofurther trigger emissions from the cell(s) to thereby cascade theeffects of the emissions to other similar or different cell typesadjacent thereto, in essentially a triggered entry into the naturalemissions process described above, preferably where the medium throughwhich the emissions are communicated is water-based, most preferablywhere the medium is the water contained within and surrounding thecells.

A further embodiment of the present invention combines the treatment ofa condition, disease or disorder with the generation of heat in theaffected target structure in order to enhance the effect of thetreatment. For example, in the treatment of a cell proliferationdisorder using a photoactivatable pharmaceutical agent (such as apsoralen or derivative thereof), one can activate the photoactivatablepharmaceutical agent by applying an initiation energy which, directly orindirectly, activates the pharmaceutical agent. As noted elsewhere inthe present application, this initiation energy can be of any type, solong as it can be converted to energy suitable for activating thepharmaceutical compound. In addition to applying this initiation energy,in this embodiment of the present invention, energy is applied thatcauses heating of the target structure. In the case of a cellproliferation disorder such as cancer, the heating would increase theproliferation rate of the cancer cells. While this may seemcounterintuitive at first, when the cell proliferation disorder is beingtreated using a DNA intercalation agent, such as psoralen or aderivative thereof, this increase in cell proliferation can actuallyassist the psoralen in causing apoptosis. In particular, when psoralenbecomes intercalated into DNA, apoptosis occurs when the cell goesthrough its next division cycle. By increasing the rate at which thecells divide, one can use the present invention methods to enhance theonset of apoptosis.

For this embodiment, the heat can be generated in any desired manner.Preferably, the heat can be generated using the application ofmicrowaves or NIR energy to the target structure or by the use of use ofnanoparticles of metal or having metal shells. In the nanoparticlesembodiment, as is done in tumor thermotherapy, magnetic metalnanoparticles can be targeted to cancer cells using conventionaltechniques, then used to generate heat by application of a magneticfield to the subject under controlled conditions. (DeNardo S J, DeNardoG L, Natarajan A et al.: Thermal dosimetry predictive of efficacy of111In-ChL6 NPAMF-induced thermoablative therapy for human breast cancerin mice. J. Nucl. Med. 48(3), 437-444 (2007).)

Alternatively, one can generate heat through the application of NIR tonanoparticles having metal shells which is converted into thermalenergy. (Hirsch L R, Stafford R J, Bankson J et al.: Nanoshell-mediatednear-infrared thermal therapy of tumors under magnetic resonanceguidance. Proc. Natl Acad. Sci. USA 100(23), 13549-13554 (2003)).

In one embodiment, the source of the initiation energy can be aradiowave emitting nanotube, such as those described by K. Jensen, J.Weldon, H. Garcia, and A. Zettl in the Department of Physics at theUniversity of California at Berkeley (seehttp://socrates.berkeley.edu/˜argon/nanoradio/radio.html, the entirecontents of which are hereby incorporated by reference). These nanotubescan be administered to the subject, and preferably would be coupled tothe activatable pharmaceutical agent or the energy modulation agent, orboth, or be located in proximity of a target cell such that uponapplication of the initiation energy, the nanotubes would accept theinitiation energy (preferably radiowaves), then emit radiowaves in closeproximity to the activatable pharmaceutical agent, or in close proximityto the energy modulation agent, or to the target cell to then cause thepredetermined cellular changes or activation of the activatablepharmaceutical agent. In such an embodiment, the nanotubes would actessentially as a radiowave focusing or amplification device in closeproximity to the activatable pharmaceutical agent or energy modulationagent or the target cell.

In one embodiment, a method for modifying a target structure whichmediates or is associated with a biological activity, comprises:

placing in a vicinity of a target structure in a subject in need oftreatment an agent receptive to microwave radiation or radiofrequencyradiation; and

applying as an initiation energy said microwave radiation orradiofrequency radiation by which the agent directly or indirectlygenerates emitted light in the infrared, visible, or ultraviolet range,wherein the emitted light contacts the target structure and induces apredetermined change in said target structure in situ,

wherein said predetermined change modifies the target structure andmodulates the biological activity of the target structure.

The initiation energy can be applied from (i) an external to the subjectinitiation energy source; or (ii) an internal to the subject initiationenergy source which is placed internally into the subject and/or thetarget structure.

Alternatively, the energy emitting source may be an energy modulationagent that emits energy in a form suitable for absorption by thetransfer agent or a target cell. For example, the initiation energysource may be acoustic energy and one energy modulation agent may becapable of receiving acoustic energy and emitting photonic energy (e.g.sonoluminescent molecules) to be received by another energy modulationagent that is capable of receiving photonic energy. Other examplesinclude transfer agents that receive energy at x-ray wavelength and emitenergy at UV wavelength, preferably at UV-A wavelength. As noted above,a plurality of such energy modulation agents may be used to form acascade to transfer energy from initiation energy source via a series ofenergy modulation agents to activate the activatable agent or thepredetermined cellular change.

In one preferred embodiment, the initiation energy source can be achemical energy source. The chemical energy source can be a memberselected from the group consisting of phosphorescent compounds,chemiluminescent compounds, bioluminescent compounds, and light emittingenzymes.

Signal transduction schemes as a drug delivery vehicle may beadvantageously developed by careful modeling of the cascade eventscoupled with metabolic pathway knowledge to sequentially orsimultaneously cause the predetermined cellular change or activatemultiple activatable pharmaceutical agents to achieve multiple-pointalterations in cellular function.

Photoactivatable agents may be stimulated by an energy source, such asirradiation, resonance energy transfer, exciton migration, electroninjection, or chemical reaction, to an activated energy state that iscapable of effecting the predetermined cellular change desired. In apreferred embodiment, the photoactivatable agent, upon activation, bindsto DNA or RNA or other structures in a cell. The activated energy stateof the agent is capable of causing damage to cells, inducing apoptosis.

One preferred method for modifying a target structure which mediates oris associated with a biological activity, comprises:

placing a nanoparticle in a vicinity of a target structure in a subjectin need of treatment, wherein the nanoparticle is configured, uponexposure to a first wavelength λ₁, to generate a second wavelength λ₂ ofradiation having a higher energy than the first wavelength λ₁, wherein

-   -   the nanoparticle comprises a metallic structure deposited in        relation to the nanoparticle,    -   a radial dimension of the metallic structure is set to a value        so that a surface plasmon resonance in the metallic structure        resonates at a frequency which provides spectral overlap with at        least one of the first wavelength λ₁ and the second wavelength        λ₂, and    -   the nanoparticle is configured to emit energy upon interaction        with an initiation energy having an energy in the range of λ₁;

administering to the subject (a) at least one activatable pharmaceuticalagent that is capable of effecting a predetermined change in the targetstructure when activated, optionally, in the presence of (b) at leastone energy modulation agent, wherein if the at least one energymodulation agent present:

-   -   (i) the at least one energy modulation agent converts the        initiation energy into a reemitted energy in the range of the        first wavelength λ₁ that causes the nanoparticle to emit energy        in the range of the second wavelength λ₂ that is capable of        activating the at least one activatable pharmaceutical agent in        situ, and/or    -   (ii) said nanoparticle upconverts the initiation energy to        generate an energy in the range of the second wavelength λ₂ that        is converted by the at least one energy modulation agent, and an        energy reemitted by the energy modulation agent is capable of        activating the at least one activatable pharmaceutical agent,

applying the initiation energy including said first wavelength λ₁ froman initiation energy source to the subject,

-   -   wherein the energy emitted by the nanoparticle including said        second wavelength λ₂ directly or indirectly activates the        activatable pharmaceutical agent in situ,    -   thus causing the predetermined change to the target structure to        occur, wherein said predetermined change modifies the target        structure and modulates the biological activity of the target        structure.

In one preferred embodiment, at least one energy modulation agent is aplurality of the energy modulation agents, and (i) the initiation energyis converted, through a cascade energy transfer between the plurality ofthe energy modulation agents, to an energy that causes the nanoparticleto emit the energy capable of activating the at least one activatablepharmaceutical agent, and/or (ii) the energy emitted by the nanoparticleis converted, through a cascade energy transfer between the plurality ofthe energy modulation agents, to an energy capable of activating the atleast one activatable pharmaceutical agent.

In one preferred embodiment, at least one activatable pharmaceuticalagent comprises an active agent contained within a photocage, whereinupon exposure to the initiation energy source, an energy emitted by thenanoparticle and/or an energy reemitted by the at least one energymodulation agent, the photocage disassociates from the active agent,rendering the active agent available.

Another preferred method for modifying a target structure which mediatesor is associated with a biological activity, comprises:

-   -   a. modifying one or more cells to incorporate a photon emitting        modification or substance;    -   b. inserting the modified cells at a targeted site of the        subject;    -   c. placing in a vicinity of a target structure in a subject in        need of treatment a nanoparticle, the nanoparticle is        configured, upon exposure to the photons emitted from the        modified cells having a first wavelength λ₁, to generate a        second wavelength λ₂ of radiation having a higher energy than        the first wavelength λ₁, wherein        -   the nanoparticle includes a metallic structure deposited in            relation to the nanoparticle,        -   a radial dimension of the metallic structure is set to a            value so that a surface plasmon resonance in the metallic            structure resonates at a frequency which provides spectral            overlap with at least one of the first wavelength λ₁ and the            second wavelength λ₂, and        -   the nanoparticle is configured to emit energy upon            interaction with an initiation energy having an energy in            the range of λ₁;    -   d. administering (i) at least one activatable pharmaceutical        agent capable of being activated directly or indirectly by the        energy emitted by the nanoparticle to cause a predetermined        change to the target structure in situ, and (ii) optionally, at        least one energy modulation agent,        -   thus causing the predetermined change to the target            structure to occur, wherein said predetermined change            modifies the target structure and modulates the biological            activity of the target structure.

In one embodiment, one or more cells are subject's own cells that havebeen removed prior to said modifying. In another embodiment, the photonemitting modification or substance is a member selected from the groupconsisting of light emitting genes; phosphorescent compounds,chemiluminescent compounds, bioluminescent compounds and light emittingenzymes.

The concept of multi-photon excitation is based on the idea that two ormore photons of low energy can excite a fluorophore in a quantum event,resulting in the emission of a fluorescence photon, typically at ahigher energy than the two or more excitatory photons. This concept wasfirst described by Maria Göppert-Mayer in her 1931 doctoraldissertation. However, the probability of the near-simultaneousabsorption of two or more photons is extremely low. Therefore a highflux of excitation photons is typically required, usually a femtosecondlaser. This had limited the range of practical applications for theconcept.

Perhaps the most well-known application of the multi-photon excitationconcept is the two-photon microscopy pioneered by Winfried Denk in thelab of Watt W. Webb at Cornell University. He combined the idea oftwo-photon absorption with the use of a laser scanner.

There is an important difference between “sequential” and “simultaneous”two photon excitation. In sequential two-photon excitation to a higherallowed energy level, the individual energies of both the first photonand the second photon must be appropriate to promote the moleculedirectly to the second allowed electronic energy level and the thirdallowed electronic energy level. In contrast, simultaneous two-photonexcitation requires only that the combined energy of the first of twophotons and the second of two photons be sufficient to promote themolecule to a second allowed electronic energy level.

In two-photon excitation microscopy, an infrared laser beam is focusedthrough an objective lens. The Ti-sapphire laser normally used has apulse width of approximately 100 femtoseconds and a repetition rate ofabout 80 MHz, allowing the high photon density and flux required for twophotons absorption and is tunable across a wide range of wavelengths.Two-photon technology is patented by Winfried Denk, James Strickler andWatt Webb at Cornell University.

Two known applications are two-photon excited fluorescence (TPEF) andnon-linear transmission (NLT). The most commonly used fluorophores haveexcitation spectra in the 400-500 nm range, whereas the laser used toexcite the fluorophores lies in the ˜700-1000 nm (infrared) range. Ifthe fluorophore absorbs two infrared photons simultaneously, it willabsorb enough energy to be raised into the excited state. Thefluorophore will then emit a single photon with a wavelength thatdepends on the type of fluorophore used (typically in the visiblespectrum). Because two photons need to be absorbed to excite afluorophore, the probability of emission is related to the intensitysquared of the excitation beam. Therefore, much more two-photonfluorescence is generated where the laser beam is tightly focused thanwhere it is more diffuse. Effectively, fluorescence is observed in anyappreciable amount in the focal volume, resulting in a high degree ofrejection of out-of-focus objects. The fluorescence from the sample isthen collected by a high-sensitivity detector, such as a photomultipliertube. This observed light intensity becomes one pixel in the eventualimage; the focal point is scanned throughout a desired region of thesample to form all the pixels of the image. Two-photon absorption can bemeasured by several techniques.

Accordingly, in one aspect, the radiative signal may be of the exactenergy required to active the photoactive agent. In this aspect, theradiative energy may be directly targeted at the desired coordinate orregion where the photoactive agent is present. The initiation energysource in this embodiment may be, for example, x-rays, gamma rays, anelectron beam, microwaves or radio waves.

In another aspect, the radiative signal may be of a lower energy thanthe excitation energy of the photoactive agent. In this aspect, theradiative signal does not have sufficient energy to activate thephotoactive agent in a conventional way. Activation of the photoactiveagent may be achieved via an “energy upgrade” mechanism such as themulti-photon mechanism described above. Activation of the photoactiveagent may further be mediated by an intermediary energy transformationagent. For example, the radiative energy may first excite a fluorophorethat emits a photon at the right energy that excites the photoactiveagent. The signal is delivered to the target photoactive agent by way ofthis intermediary agent. In this way, in addition to energy upgrading(and downgrading, as described below), a signal relay mechanism is alsointroduced. The initiation energy source may be x-rays, gamma rays, anelectron beam, microwaves or radio waves. In one embodiment, the energyupgrades are obtained via 2, 3, 4, or 5 photon absorptions.

Work in the area of photodynamic therapy has shown that the amount ofsinglet oxygen required to cause cell lysis, and thus cell death, is0.32×10⁻³ mol/liter or more, or 10⁹ singlet oxygen molecules/cell ormore. In one preferred embodiment, it is preferable to avoid productionof an amount of singlet oxygen that would cause cell lysis, due to itsindiscriminate nature of attack, lysing both target cells and healthycells. Accordingly, it is preferred in one preferred embodiment that thelevel of singlet oxygen production caused by the initiation energy usedor activatable pharmaceutical agent upon activation be less than levelneeded to cause cell lysis.

One advantage is that multiple wavelengths of emitted radiation may beused to selectively stimulate one or more photoactivatable agents orenergy modulation agents capable of stimulating the one or morephotoactivatable agents. The energy modulation agent is preferablystimulated at a wavelength and energy that causes little or no damage tohealthy cells, with the energy from one or more energy modulation agentsbeing transferred, such as by Foerster Resonance Energy Transfer, to thephotoactivatable agents that damage the cell and cause the onset of thedesired cellular change, e.g., apoptosis of the cells.

Another advantage is that side effects can be greatly reduced bylimiting the production of free radicals, singlet oxygen, hydroxides andother highly reactive groups that are known to damage healthy cells.Furthermore, additional additives, such as antioxidants, may be used tofurther reduce undesired effects of irradiation.

Resonance Energy Transfer (RET) is an energy transfer mechanism betweentwo molecules having overlapping emission and absorption bands.Electromagnetic emitters are capable of converting an arrivingwavelength to a longer wavelength. For example, UV-B energy absorbed bya first molecule may be transferred by a dipole-dipole interaction to aUV-A-emitting molecule in close proximity to the UV-B-absorbingmolecule. Alternatively, a material absorbing a shorter wavelength maybe chosen to provide RET to a non-emitting molecule that has anoverlapping absorption band with the transferring molecule's emissionband. Alternatively, phosphorescence, chemiluminescence, orbioluminescence may be used to transfer energy to a photoactivatablemolecule.

Alternatively, one can administer the initiation energy source to thesubject. Within the context of the present invention, the administeringof the initiation energy source means the administration of an agent,that itself produces the initiation energy, in a manner that permits theagent to arrive at the target cell within the subject without beingsurgically inserted into the subject. The administration can take anyform, including, but not limited to, oral, intravenous, intraperitoneal,inhalation, etc. Further, the initiation energy source in thisembodiment can be in any form, including, but not limited to, tablet,powder, liquid solution, liquid suspension, liquid dispersion, gas orvapor, etc. In this embodiment, the initiation energy source includes,but is not limited to, chemical energy sources, nanoemitters, nanochips,and other nanomachines that produce and emit energy of a desiredfrequency. Recent advances in nanotechnology have provided examples ofvarious devices that are nanoscale and produce or emit energy, such asthe Molecular Switch (or Mol-Switch) work by Dr. Keith Firman of the ECResearch and Development Project, or the work of Cornell et al. (1997)who describe the construction of nanomachines based around ion-channelswitches only 1.5 nm in size, which use ion channels formed in anartificial membrane by two gramicidin molecules: one in the lower layerof the membrane attached to a gold electrode and one in the upper layertethered to biological receptors such as antibodies or nucleotides. Whenthe receptor captures a target molecule or cell, the ion channel isbroken, its conductivity drops, and the biochemical signal is convertedinto an electrical signal. These nanodevices could also be coupled withthe present invention to provide targeting of the target cell, todeliver the initiation energy source directly at the desired site.

In another embodiment, the present invention, includes theadministration of a source of chemical energy such as chemiluminescence,phosphorescence or bioluminescence. The source of chemical energy can bea chemical reaction between two or more compounds, or can be induced byactivating a chemiluminescent, phosphorescent or bioluminescent compoundwith an appropriate activation energy, either outside the subject orinside the subject, with the chemiluminescence, phosphorescence orbioluminescence being allowed to activate the activatable pharmaceuticalagent in vive after administration. In one embodiment, the activatablepharmaceutical agent and the source of chemical energy can beadministered. The administration can be performed sequentially in anyorder or simultaneously. In the case of certain sources of such chemicalenergy, the administration of the chemical energy source can beperformed after activation outside the subject, with the lifetime of theemission of the energy being up to several hours for certain types ofphosphorescent materials for example. There are no known previousefforts to use resonance energy transfer of any kind to activate anintercalator to bind DNA.

Yet another example is that nanoparticles or nanoclusters of certainatoms may be introduced such that are capable of resonance energytransfer over comparatively large distances, such as greater than onenanometer, more preferably greater than five nanometers, even morepreferably at least 10 nanometers. Functionally, resonance energytransfer may have a large enough “Foerster” distance (R₀), such thatnanoparticles in one part of a cell are capable of stimulatingactivation of photoactivatable agents disposed in a distant portion ofthe cell, so long as the distance does not greatly exceed R₀. Forexample, gold nanospheres having a size of 5 atoms of gold have beenshown to have an emission band in the ultraviolet range, recently.

In one embodiment, an aggressive cell proliferation disorder has a muchhigher rate of mitosis, which leads to selective destruction of adisproportionate share of the malignant cells during even a systemicallyadministered treatment. Stem cells and healthy cells may be spared fromwholesale programmed cell death, even if exposed to photoactivatedagents, provided that such photoactivated agents degenerate from theexcited state to a lower energy state prior to binding, mitosis or othermechanisms for creating damage to the cells of a substantial fraction ofthe healthy stem cells. Thus, an auto-immune response may not beinduced.

Alternatively, a blocking agent may be used that prevents or reducesdamage to stem cells or healthy cells, selectively, which wouldotherwise be impaired. The blocking agent is selected or is administeredsuch that the blocking agent does not impart a similar benefit tomalignant cells, for example.

In one embodiment, stem cells are targeted, specifically, fordestruction with the intention of replacing the stem cells with a donorcell line or previously stored, healthy cells of the patient. In thiscase, no blocking agent is used. Instead, a carrier or photosensitizeris used that specifically targets the stem cells.

Any of the photoactivatable agents may be exposed to an excitationenergy source implanted in a subject preferably near a target site. Thephotoactive agent may be directed to a receptor site by a carrier havinga strong affinity for the receptor site. Within the context of thepresent invention, a “strong affinity” is preferably an affinity havingan equilibrium dissociation constant, K_(i), at least in the nanomolar,nM, range or higher. Preferably, the carrier may be a polypeptide andmay form a covalent bond with a photoactive agent, for example. Thepolypeptide may be an insulin, interleukin, thymopoietin or transferrin,for example. Alternatively, a photoactive agent may have a strongaffinity for the target cell without binding to a carrier.

A receptor site may be any of the following: nucleic acids of nucleatedblood cells, molecule receptor sites of nucleated blood cells, theantigenic sites on nucleated blood cells, epitopes, or other sites wherephotoactive agents are capable of destroying a targeted cell.

In one embodiment, thin fiber optic lines are inserted in the subjectand laser light is used to photoactivate the agents. In anotherembodiment, a plurality of sources for supplying electromagneticradiation energy or energy transfer is provided by one or more moleculesadministered to a patient. The molecules may emit stimulating radiationin the correct band of wavelength to stimulate the target structuredirectly or to simulate the photoactivatable agents, or the moleculesmay transfer energy by a resonance energy transfer or other mechanismdirectly to the target structure or the photoactivatable agent orindirectly by a cascade effect via other molecular interactions.

In another embodiment, the patient's own cells are removed andgenetically modified to provide photonic emissions. For example, tumoror healthy cells may be removed, genetically modified to inducebioluminescence and may be reinserted at the site of the disease orcondition to be treated. The modified, bioluminescent cells may befurther modified to prevent further division of the cells or division ofthe cells only so long as a regulating agent is present.

In a further embodiment, a biocompatible emitting source, such as afluorescing metal nanoparticle or fluorescing dye molecule, is selectedthat emits in the UV-A band. The UV-A emitting source is directed to thesite of a disease or condition. The UV-A emitting source may be directedto the site of the disease or condition by systemically administeringthe UV-A emitting source. Preferably, the UV-A emitting source isconcentrated in the target site, such as by physical insertion or byconjugating the UV-A emitting molecule with a specific carrier that iscapable of concentrating the UV-A emitting source in a specific targetstructure, as is known in the art.

In one preferred embodiment, the UV-A emitting source is a goldnanoparticle comprising a cluster of 5 gold atoms, such as a watersoluble quantum dot encapsulated by polyamidoamine dendrimers. The goldatom clusters may be produced through a slow reduction of gold salts(e.g. HAuCl₄ or AuBr₃) or other encapsulating amines, for example. Oneadvantage of such a gold nanoparticle is the increased Foerster distance(i.e. R₀), which may be greater than 100 angstroms. The equation fordetermining the Foerster distance is substantially different from thatfor molecular fluorescence, which is limited to use at distances lessthan 100 angstroms. It is believed that the gold nanoparticles aregoverned by nanoparticle surface to dipole equations with a 1/R⁴distance dependence rather than a 1/R⁶ distance dependence. For example,this permits cytoplasmic to nuclear energy transfer between metalnanoparticles and a photoactivatable molecule, such as a psoralen andmore preferably an 8-methoxypsoralen (8-MOP) administered orally to apatient, which is known to be safe and effective at inducing anapoptosis of leukocytes.

In another embodiment, a UV- or light-emitting luciferase is selected asthe emitting source for exciting a photoactivatable agent. A luciferasemay be combined with ATP or another molecule, which may then beoxygenated with additional molecules to stimulate light emission at adesired wavelength. Alternatively, a phosphorescent emitting source maybe used. One advantage of a phosphorescent emitting source is that thephosphorescent emitting molecules or other source may beelectroactivated or photoactivated prior to insertion into a target siteeither by systemic administration or direct insertion into the region ofthe target site. Phosphorescent materials may have longer relaxationtimes than fluorescent materials, because relaxation of a triplet stateis subject to forbidden energy state transitions, storing the energy inthe excited triplet state with only a limited number of quantummechanical energy transfer processes available for returning to thelower energy state. Energy emission is delayed or prolonged from afraction of a second to several hours. Otherwise, the energy emittedduring phosphorescent relaxation is not otherwise different thanfluorescence, and the range of wavelengths may be selected by choosing aparticular phosphor.

In another embodiment, a combined electromagnetic energy harvestermolecule is designed, such as the combined light harvester disclosed inJ. Am. Chem. Soc. 2005, 127, 9760-9768, the entire contents of which arehereby incorporated by reference. By combining a group of fluorescentmolecules in a molecular structure, a resonance energy transfer cascademay be used to harvest a wide band of electromagnetic radiationresulting in emission of a narrow band of fluorescent energy. By pairinga combined energy harvester with a photoactivatable molecule, a furtherenergy resonance transfer excites the photoactivatable molecule, whenthe photoactivatable molecule is nearby stimulated combined energyharvester molecules. Another example of a harvester molecule isdisclosed in FIG. 4 of “Singlet-Singlet and Triplet-Triplet EnergyTransfer in Bichromophoric Cyclic Peptides,” M.S. Thesis by M. O. Guler,Worcester Polytechnic Institute, May 18, 2002, which is incorporatedherein by reference.

In another embodiment, a Stokes shift of an emitting source or a seriesof emitting sources arranged in a cascade is selected to convert ashorter wavelength energy, such as X-rays, to a longer wavelengthfluorescence emission such a optical or UV-A, which is used to stimulatea photoactivatable molecule at the location of the target structure.Preferably, the photoactivatable molecule is selected to cause thepredetermined change in target structure without causing substantialharm to normal, healthy cells.

In an additional embodiment, the photoactivatable agent can be aphotocaged complex having an active agent contained within a photocage.The active agent is bulked up with other molecules that prevent it frombinding to specific targets, thus masking its activity. When thephotocage complex is photoactivated, the bulk falls off, exposing theactive agent. In such a photocage complex, the photocage molecules canbe photoactive (i.e. when photoactivated, they are caused to dissociatefrom the photocage complex, thus exposing the active agent within), orthe active agent can be the photoactivatable agent (which whenphotoactivated causes the photocage to fall off), or both the photocageand the active agent are photoactivated, with the same or differentwavelengths. For example, a toxic chemotherapeutic agent can bephotocaged, which will reduce the systemic toxicity when delivered. Oncethe agent is concentrated in the tumor, the agent is irradiated with anactivation energy. This causes the “cage” to fall off, leaving acytotoxic agent in the tumor cell. Suitable photocages include thosedisclosed by Young and Deiters in “Photochemical Control of BiologicalProcesses”, Org. Biomol. Chem., 5, pp. 999-1005 (2007) and“Photochemical Hammerhead Ribozyme Activation”, Bioorganic & MedicinalChemistry Letters, 16(10), pp. 2658-2661 (2006), the contents of whichare hereby incorporated by reference.

In one preferred embodiment, the use of light for uncaging a compound oragent is used for elucidation of neuron functions and imaging, forexample, two-photon glutamine uncaging (Harvey C D, et al., Nature,450:1195-1202 (2007); Eder M, et al., Rev. Neurosci., 15:167-183(2004)). Other signaling molecules can be released by UV lightstimulation, e.g., GABA, secondary messengers (e.g., Ca²⁺ and Mg²⁺),carbachol, capsaicin, and ATP (Zhang F., et al., 2006). Chemicalmodifications of ion channels and receptors may be carried out to renderthem light-responsive. Ca²⁺ is involved in controlling fertilization,differentiation, proliferation, apoptosis, synaptic plasticity, memory,and developing axons. In yet another preferred embodiment, Ca²⁺ wavescan be induced by UV irradiation (single-photon absorption) and NIRirradiation (two-photon absorption) by releasing caged Ca²⁺, anextracellular purinergic messenger InsP3 (Braet K., et al., CellCalcium, 33:37-48 (2003)), or ion channel ligands (Zhang F., et al.,2006).

Genetic targeting allows morphologically and electrophysipologicallycharacterization of genetically defined cell populations. Accordingly,in an additional embodiment, a light-sensitive protein is introducedinto cells or live subjects via a number of techniques includingelectroporation, DNA microinjection, viral delivery, liposomaltransfection, creation of transgenic lines and calcium-phosphateprecipitation. For example, lentiviral technology provides a convenientcombination a conventional combination of stable long-term expression,ease of high-titer vector production and low immunogenicity. Thelight-sensitive protein may be, for example, channelrhodopsin-2 (ChR2)and chloride pump halorhodopsin (NpHR). The light protein encodinggene(s) along with a cell-specific promoter can be incorporated into thelentiviral vector or other vector providing delivery of thelight-sensitive protein encoding gene into a target cell. ChR2containing a light sensor and a cation channel, provides electricalstimulation of appropriate speed and magnitude to activate neuronalspike firing, when the cells harboring Ch2R are pulsed with light.

In one embodiment, a lanthanide chelate capable of intense luminescenceis used. For example, a lanthanide chelator may be covalently joined toa coumarin or coumarin derivative or a quinolone or quinolone-derivativesensitizer. Sensitizers may be a 2- or 4-quinolone, a 2- or 4-coumarin,or derivatives or combinations of these examples. A carbostyril 124(7-amino-4-methyl-2-quinolone), a coumarin 120(7-amino-4-methyl-2-coumarin), a coumarin 124(7-amino-4-(trifluoromethyl)-2-coumarin), aminoinethyltrimethylpsoralenor other similar sensitizer may be used. Chelates may be selected toform high affinity complexes with lanthanides, such as terbium oreuropium, through chelator groups, such as DTPA. Such chelates may becoupled to any of a wide variety of well known probes or carriers, andmay be used for resonance energy transfer to a psoralen orpsoralen-derivative, such as 8-MOP, or other photoactive moleculescapable of binding DNA. In one alternative example, the lanthanidechelate is localized at the site of the disease using an appropriatecarrier molecule, particle or polymer, and a source of electromagneticenergy is introduced by minimally invasive procedures to irradiate thetarget structure, after exposure to the lanthanide chelate and aphotoactive molecule.

In another embodiment, a biocompatible, endogenous fluorophore emitteris selected to stimulate resonance energy transfer to a photoactivatablemolecule. A biocompatible emitter with an emission maxima within theabsorption range of the biocompatible, endogenous fluorophore emittermay be selected to stimulate an excited state in fluorophore emitter,One or more halogen atoms may be added to any cyclic ring structurecapable of intercalation between the stacked nucleotide bases in anucleic acid (either DNA or RNA) to confer new photoactive properties tothe intercalator. Any intercalating molecule (psoralens, coumarins, orother polycyclic ring structures) may be selectively modified byhalogenation or addition of non-hydrogen bonding ionic substituents toimpart advantages in its reaction photochemistry and its competitivebinding affinity for nucleic acids over cell membranes or chargedproteins, as is known in the art.

Skin photosensitivity is a major toxicity of photosensitizers. Severesunburn occurs if skin is exposed to direct sunlight for even a fewminutes. Early marine research hinted at a vigorous and long termstimulation of immune response; however, actual clinical testing hasfailed to achieve the early promises of photodynamic therapies. Theearly photosensitizers for photodynamic therapies targeted type IIresponses, which created singlet oxygen when photoactivated in thepresence of oxygen. The singlet oxygen caused cellular necrosis and wasassociated with inflammation and an immune response. Some additionalphotosensitizers have been developed to induce type I responses,directly damaging cellular structures, Porfimer sodium (Photofrin; QLTTherapeutics, Vancouver, BC, Canada), is a partially purifiedpreparation of hematoporphyrin derivative (HpD). Photofrin has beenapproved by the US Food and Drug Administration for the treatment ofobstructing esophageal cancer, microinvasive endobronchial non-smallcell lung cancer, and obstructing endobronchial non-small cell lungcancer. Photofrin is activated with 630 nm, which has a tissuepenetration of approximately 2 to 5 mm. Photofrin has a relatively longduration of skin photosensitivity (approximately 4 to 6 weeks).

Tetra (m-hydroxyphenyl) chlorin (Foscan; Scotia Pharmaceuticals,Stirling, UK), is a synthetic chlorine compound that is activated by 652nm light. Clinical studies have demonstrated a tissue effect of up to 10mm with Foscan and 652 nm light. Foscan is more selectively aphotosensitizer in tumors than normal tissues, and requires acomparatively short light activation time. A recommended dose of 0.1mg/kg is comparatively low and comparatively low doses of light may beused. Nevertheless, duration of skin photosensitivity is reasonable(approximately 2 weeks). However, Foscan induces a comparatively highyield of singlet oxygen, which may be the primary mechanism of DNAdamage for this molecule.

Motexafin lutetium (Lutetium texaphryin) is activated by light in thenear infrared region (732 nm). Absorption at this wavelength has theadvantage of potentially deeper penetration into tissues, compared withthe amount of light used to activate other photosensitizers (FIGS. 2Aand 2B). Lutetium texaphryin also has one of the greatest reportedselectivities for tumors compared to selectivities of normal tissues.Young S W, et al.: Lutetium texaphyrin (PCI-0123) a near-infrared,water-soluble photosensitizer. Photochem Photobiol 1996, 63:892-897. Inaddition, its clinical use is associated with a shorter duration of skinphotosensitivity (24 to 48 hours). Lutetium texaphryin has beenevaluated for metastatic skin cancers. It is currently underinvestigation for treatment of recurrent breast cancer and for locallyrecurrent prostate cancer. The high selectivity for tumors promisesimproved results in clinical trials.

In general, the approach may be used with any source for the excitationof higher electronic energy states, such as electrical, chemical and/orradiation, individually or combined into a system for activating anactivatable molecule. The process may be a photopheresis process or maybe similar to photophoresis. While photophoresis is generally thought tobe limited to photonic excitation, such as by UV-light, other forms ofradiation may be used as a part of a system to activate an activatablemolecule. Radiation includes ionizing radiation which is high energyradiation, such as an X-ray or a gamma ray, which interacts to produceion pairs in matter. Radiation also includes high linear energy transferirradiation, low linear energy transfer irradiation, alpha rays, betarays, neutron beams, accelerated electron beams, and ultraviolet rays.Radiation also includes proton, photon and fission-spectrum neutrons.Higher energy ionizing radiation may be combined with chemical processesto produce energy states favorable for resonance energy transfer, forexample. Other combinations and variations of these sources ofexcitation energy may be combined as is known in the art, in order tostimulate the activation of an activatable molecule, such as 8-MOP. Inone example, ionizing radiation is directed at a solid tumor andstimulates, directly or indirectly, activation of 8-MOP, as well asdirectly damaging the DNA of malignant tumor cells. In this example,either the effect of ionizing radiation or the photophoresis-likeactivation of 8-MOP may be thought of as an adjuvant therapy to theother.

Work in the area of photodynamic therapy has shown that the amount ofsinglet oxygen required to cause cell lysis, and thus cell death, is0.32×10⁻³ mol/liter or more, or 10⁹ singlet oxygen molecules/cell ormore. However, in the present invention, it is most preferable to avoidproduction of an amount of singlet oxygen that would cause cell lysis,due to its indiscriminate nature of attack, lysing both target cells andhealthy cells. Accordingly, it is most preferred in the presentinvention that the level of singlet oxygen production caused by theinitiation energy used or activatable pharmaceutical agent uponactivation be less than level needed to cause cell lysis.

In yet another embodiment, the activatable pharmaceutical agent,preferably a photoactive agent, is directed to a receptor site by acarrier having a strong affinity for the receptor site. The carrier maybe a polypeptide and may form a covalent bond with a photo active agent,for example. The polypeptide may be an insulin, interleukin,thymopoietin or transferrin, for example. Alternatively, a photoactivepharmaceutical agent may have a strong affinity for the target cellwithout a binding to a carrier.

For example, a treatment may be applied that acts to slow or pausemitosis. Such a treatment is capable of slowing the division of rapidlydividing healthy cells or stem cells without pausing mitosis ofcancerous cells. Thus, the difference in growth rate between thenon-target cells and target cells are further differentiated to enhancethe effectiveness of the methods of the present invention.

In a further embodiment, methods in accordance with the presentinvention may further include adding an additive to alleviate treatmentside-effects, Exemplary additives may include, but are not limited to,antioxidants, adjuvant, or combinations thereof. In one exemplaryembodiment, psoralen is used as the activatable pharmaceutical agent,UV-A is used as the activating energy, and antioxidants are added toreduce the unwanted side-effects of irradiation.

In another aspect, the present invention also provides methods forproducing an autovaccine, comprising:

(1) providing a population of target cells;

(2) pacing in a vicinity of a target structure in the target cells ananoparticle, the nanoparticle is configured, upon exposure to a firstwavelength λ₁, to generate a second wavelength λ₂ of radiation having ahigher energy than the first wavelength λ₁, wherein

-   -   the nanoparticle comprises a metallic structure deposited in        relation to the nanoparticle,    -   a radial dimension of the metallic structure is set to a value        so that a surface plasmon resonance in the metallic structure        resonates at a frequency which provides spectral overlap with at        least one the first wavelength λ₁ and the second wavelength λ₂,        and    -   the nanoparticle is configured to emit energy in the vicinity of        or into the target structure upon interaction with an initiation        energy having an energy in the range of λ₁;

(3) treating the target cells ex vivo in an environment separate andisolated from the subject with a psoralen or a derivative thereof;

(4) applying the initiation energy from an initiation energy sourceincluding said first wavelength λ₁ to the target cells ex vivo, whereinthe emitted energy including said second wavelength λ₂ directly orindirectly contacts the target structure and induces a predeterminedcellular change in the target cells, and, optionally, applying at leastone energy modulation agent; and

(5) returning the thus changed cells back to the subject to induce inthe subject an autovaccine effect against the target cell, wherein thechanged cells act as an autovaccine.

In a different embodiment, the predetermined change enhances theexpression of, promotes the growth of, or increases the quantity of saidtarget structure; enhances, inhibits or stabilizes the usual biologicalactivity of said target structure compared to a similar untreated targetstructure, and/or alters the immunological or chemical properties ofsaid target structure. In a different embodiment, said target structureis a compound that is modified by said predetermined change to be moreor less antigenic or immunogenic.

The activatable pharmaceutical agent and derivatives thereof as well asthe energy modulation agent, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the activatable pharmaceutical agent and a pharmaceuticallyacceptable carrier. The pharmaceutical composition also comprises atleast one additive having a complementary therapeutic or diagnosticeffect, wherein the additive is one selected from an antioxidant, anadjuvant, or a combination thereof.

One preferred pharmaceutical composition for modifying a targetstructure which mediates or is associated with a biological activity,comprises:

a nanoparticle, wherein the nanoparticle is configured, upon exposure toa first wavelength λ1, to generate a second wavelength λ2 of radiationhaving a higher energy than the first wavelength λ1,

-   -   the nanoparticle comprises a metallic structure deposited in        relation to the nanoparticle,    -   a radial dimension of the metallic structure is set to a value        so that a surface plasmon resonance in the metallic structure        resonates at a frequency which provides spectral overlap with at        least one of the first wavelength λ1 and the second wavelength        λ2, and    -   the nanoparticle is configured to emit light in the vicinity of        or into the target structure upon interaction with an initiation        energy having an energy in the range of λ1;    -   wherein the energy modulation agent, if present,        -   (i) is capable of converting the initiation energy to an            energy that causes the nanoparticle to generate an energy in            the range of the second wavelength λ2 which is capable,            directly or indirectly, of inducing a predetermined change            in the target structure with or without the activatable            pharmaceutical agent; and/or        -   (ii) said nanoparticle upconverts the initiation energy into            an energy in the range of said second wavelength λ2 that is            converted by the energy modulation agent, and an energy            reemitted by the energy modulation agent is capable of            inducing, directly or indirectly, the predetermined change            in the target structure; and

a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition can also comprise anenergy source, for example, a chemical energy source which can be amember selected from the group consisting of phosphorescent compounds,chemiluminescent compounds, bioluminescent compounds and light emittingenzymes. In another embodiment, the pharmaceutical composition comprisesat least one energy modulation agent and/or at least one activatablepharmaceutical agent.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions. Modifications can be made to the compound of thepresent invention to affect solubility or clearance of the compound.These molecules may also be synthesized with D-amino acids to increaseresistance to enzymatic degradation. If necessary, the activatablepharmaceutical agent can be co-administered with a solubilizing agent,such as cyclodextran.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, rectal administration, and direct injection into theaffected area, such as direct injection into a tumor. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerin, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates, and agents for the adjustment of tonicity suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of administering agents according to the present invention arenot limited to the conventional means such as injection or oralinfusion, but include more advanced and complex forms of energytransfer. For example, genetically engineered cells that carry andexpress energy modulation agents may be used. Cells from the host may betransfected with genetically engineered vectors that expressbioluminescent agents. Transfection may be accomplished via in situ genetherapy techniques such as injection of viral vectors or gene guns, ormay be performed ex vivo by removing a sample of the host's cells andthen returning to the host upon successful transfection.

Such transfected cells may be inserted or otherwise targeted at the sitewhere diseased cells are located. In this embodiment, the initiationenergy source may be a biochemical source as such ATP, in which case theinitiation energy source is considered to be directly implanted in thetransfected cell. Alternatively, a conventional micro-emitter devicecapable of acting as an initiation energy source may be transplanted atthe site of the diseased cells.

It will also be understood that the order of administering the differentagents is not particularly limited. Thus in some embodiments theactivatable pharmaceutical agent may be administered before the energymodulation agent, while in other embodiments the energy modulation agentmay be administered prior to the activatable pharmaceutical agent. Itwill be appreciated that different combinations of ordering may beadvantageously employed depending on factors such as the absorption rateof the agents, the localization and molecular trafficking properties ofthe agents, and other pharmacokinetics or pharmacodynamicsconsiderations.

A further embodiment is the use of the present invention for thetreatment of skin cancer. In this example, a photoactivatable agent,preferably psoralen, is given to the patient, and is delivered to theskin lesion via the blood supply. An activation source having limitedpenetration ability (such as UV or IR) is shined directly on the skin—inthe case of psoralen, it would be a UV light, or an IR source. With theuse of an IR source, the irradiation would penetrate deeper and generateUV via two single photon events with psoralen.

In a further embodiment, methods according to this aspect of the presentinvention further include a step of separating the components of thetreated cells into fractions and testing each fraction for autovaccineeffect in a host. The components thus isolated and identified may thenserve as an effective autovaccine to stimulate the host's immune systemto suppress growth of the targeted cells.

In different aspect of the invention, a kit for modifying a targetstructure which mediates or is associated with a biological activity,comprising:

a nanoparticle, wherein the nanoparticle is configured, upon exposure toa first wavelength λ₁, to generate a second wavelength λ₂ of radiationhaving a higher energy than the first wavelength λ₁,

-   -   the nanoparticle comprises a metallic structure deposited in        relation to the nanoparticle,    -   a radial dimension of the metallic structure is set to a value        so that a surface plasmon resonance in the metallic structure        resonates at a frequency which provides spectral overlap with at        least one of the first wavelength λ₁ and the second wavelength        λ₂, and    -   the nanoparticle is configured to emit light in the vicinity of        or into the target upon interaction with an initiation energy        having an energy in the range of λ₁;

optionally, an energy modulation agent, wherein the energy modulationagent, if present,

-   -   (i) converts the initiation energy to an energy in the range of        the first wavelength λ₁, to generate the second wavelength λ₂ of        radiation by the nanoparticle capable of causing, either        directly or indirectly, a predetermined change in the target        structure with or without the activatable pharmaceutical agent;        and/or    -   (ii) said nanoparticle upconverts the initiation energy to an        energy in the range of said second wavelength λ2 that is        converted by the energy modulation agent, and an energy        reemitted by the energy modulation agent causes, directly or        indirectly, the predetermined change in the target structure        with or without an activatable pharmaceutical agent; and

one or more containers suitable for storing the agents in stable forms.

In another embodiment, the kit can further comprise instructions foradministering the nanoparticle. In another embodiment, the kit comprisesat least one energy modulation agent and/or the activatablepharmaceutical agent to a subject.

In one embodiment, a system for modifying a target structure whichmediates or is associated with a biological activity, comprises:

a nanoparticle, wherein the nanoparticle is configured, upon exposure toa first wavelength λ1, to generate a second wavelength λ2 of radiationhaving a higher energy than the first wavelength λ1,

a metallic structure depossited in relation to the nanoparticle,

wherein

-   -   a radial dimension of the metallic structure is set to a value        so that a surface plasmon resonance in the metallic structure        resonates at a frequency which provides spectral overlap with at        least one of the first wavelength λ1 and the second wavelength        λ2, and    -   the nanoparticle is configured to emit energy in the vicinity of        or into the target structure upon interaction with an initiation        energy having an energy in the range of λ1;

a mechanism for placing the nanoparticle in the subject; and

an initiation energy source to provide the initiation energy capable tobe upconverted by the nanoparticle, wherein the emitted energy iscapable of inducing a predetermined change in the target structure insitu,

wherein said predetermined change is capable of modifying the targetstructure and modulating the biological activity of the targetstructure.

In another preferred embodiment, the system further comprises at leastone agent selected from the group consisting of at least one energymodulation agent, at least one activatable pharmaceutical agent and atleast one plasmonics-active agent.

In yet another embodiment, when at least one energy modulation agent ispresent in the system,

(i) the energy modulation agent is capable of converting the initiationenergy to an energy that causes the nanoparticle to generate an energyin the range of the second wavelength λ2 which is capable, directly orindirectly, of inducing a predetermined change in the target structurewith or without the activatable pharmaceutical agent; and/or

(ii) said nanoparticle is capable of upconverting the initiation energyinto an energy in the range of said second wavelength λ2 that isconverted by the energy modulation agent, and an energy reemitted by theenergy modulation agent is capable of inducing, directly or indirectly,the predetermined change in the target structure.

The reagents and chemicals useful for the present invention may bepackaged in kits to facilitate application of the present invention. Inone exemplary embodiment, a kit including a psoralen, and fractionatingcontainers for easy fractionation and isolation of autovaccines iscontemplated. A further embodiment of kit would comprise at least oneactivatable pharmaceutical agent capable of causing a predeterminedcellular change, at least one energy modulation agent capable ofactivating the at least one activatable agent when energized, at leastone plasmonics agent and containers suitable for storing the agents instable form, and preferably further comprising instructions foradministering the at least one activatable pharmaceutical agent, atleast one plasmonics agent and at least one energy modulation agent to asubject, and for applying an initiation energy from an initiation energysource to activate the activatable pharmaceutical agent. Theinstructions could be in any desired form, including but not limited to,printed on a kit insert, printed on one or more containers, as well aselectronically stored instructions provided on an electronic storagemedium, such as a computer readable storage medium. Also optionallyincluded is a software package on a computer readable storage mediumthat permits the user to integrate the information and calculate acontrol dose, to calculate and control intensity of the irradiationsource.

The activatable pharmaceutical agent and derivatives thereof as well asthe energy modulation agent and plasmonics compounds and structures, canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the activatablepharmaceutical agent and a pharmaceutically acceptable carrier. Thepharmaceutical composition also comprises at least one additive having acomplementary therapeutic or diagnostic effect, wherein the additive isone selected from an antioxidant, an adjuvant, or a combination thereof.

An advantage of the methods of the present invention is that byspecifically targeting cells affected by a cell proliferation disorder,such as rapidly dividing cells, and triggering a cellular change, suchas apoptosis, in these cells in situ, the immune system of the host maybe stimulated to have an immune response against the diseased cells.Once the host's own immune system is stimulated to have such a response,other diseased cells that are not treated by the activatablepharmaceutical agent may be recognized and be destroyed by the host'sown immune system. Such autovaccine effects may be obtained, forexample, in treatments using psoralen and UV-A.

The present invention methods can be used alone or in combination withother therapies for treatment of cell proliferation disorders,Additionally, the present invention methods can be used, if desired, inconjunction with recent advances in chronomedicine, such as thatdetailed in Giacchetti et al, Journal of Clinical Oncology, Vol 24, No22 (August 1), 2006: pp. 3562-3569. In chronomedicine it has been foundthat cells suffering from certain types of disorders, such as cancer,respond better at certain times of the day than at others. Thus,chronomedicine could be used in conjunction with the present methods inorder to augment the effect of the treatments of the present invention.

In preferred embodiments, the initiation energy source may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. One example of such linear accelerators is the SmartBeam™IMRT (intensity modulated radiation therapy) system from Varian medicalsystems (Varian Medical Systems, Inc., Palo Alto, Calif.).

In other embodiments, endoscopic or laproscopic devices equipped withappropriate initiation energy emitter may be used as the initiationenergy source. In such systems, the initiation energy may be navigatedand positioned at the pre-selected coordinate to deliver the desiredamount of initiation energy to the site.

Plasmonics Enhanced Photospectral Therapy (PEPST)

In the PEPST embodiment of the present invention, the present inventionis significantly different from the phototherapy technique oftenreferred to Photo-thermal Therapy (PTT). To illustrate the differencebetween the present invention PEPST, a form of photospectral therapy(PST) and the PTT technique, the photochemical processes involved in PSTand PPT is discussed below.

When drug molecules absorb excitation light, electrons undergotransitions from the ground state to an excited electronic state. Theelectronic excitation energy subsequently relaxes via radiative emission(luminescence) and radiationless decay channels. When a molecule absorbsexcitation energy, it is elevated from S_(o) to some vibrational levelof one of the excited singlet states, S_(m), in the manifold S₁, . . . ,S_(n). In condensed media (tissue), the molecules in the S, statedeactivate rapidly, within 10⁻¹³ to 10⁻¹¹ s via vibrational relaxation(VR) processes, ensuring that they are in the lowest vibrational levelsof S_(n) possible. Since the VR process is faster than electronictransitions, any excess vibrational energy is rapidly lost as themolecules are deactivated to lower vibronic levels of the correspondingexcited electronic state. This excess VR energy is released as thermalenergy to the surrounding medium. From the S_(n) state, the moleculedeactivates rapidly to the isoenergetic vibrational level of a lowerelectronic state such as S_(n-1) via an internal conversion (IC)process. IC processes are transitions between states of the samemultiplicity. The molecule subsequently deactivates to the lowestvibronic levels of S_(n-1) via a VR process. By a succession of ICprocesses immediately followed by VR processes, the molecule deactivatesrapidly to the ground state S₁. This process results in excess VR and ICenergy released as thermal energy to the surrounding medium leading tothe overheating of the local environment surrounding the light absorbingdrug molecules. The heat produced results in local cell or tissuedestruction. The light absorbing species include natural chromophores intissue or exogenous dye compounds such as indocyanine green,naphthalocyanines, and porphyrins coordinated with transition metals andmetallic nanoparticles and nanoshells of metals. Natural chromophores,however, suffer from very low absorption. The choice of the exogenousphotothermal agents is made on the basis of their strong absorptioncross sections and highly efficient light-to-heat conversion. Thisfeature greatly minimizes the amount of laser energy needed to inducelocal damage of the diseased cells, making the therapy method lessinvasive. A problem associated with the use of dye molecules is theirphotobleaching under laser irradiation. Therefore, nanoparticles such asgold nanoparticles and nanoshells have recently been used. The promisingrole of nanoshells in photothermal therapy of tumors has beendemonstrated [Hirsch, L. R., Stafford, R. J., Bankson, J. A., Sershen,S. R., Rivera, B., Price, R. E., Hazle, J. D., Halas, N. J., and West,J. L., Nanoshell-mediated near-infrared thermal therapy of tumors undermagnetic resonance guidance. PNAS, 2003. 100(23): p. 13549-13554]. Theuse of plasmonics-enhanced photothermal properties of metalnanoparticles for photothermal therapy has also been reviewed (XiaohuaHuang & Prashant K. Jain & Ivan H. El-Sayed & Mostafa A. El-Sayed,“Plasmonic photothermal therapy (PPTT) using gold nanoparticles,” Lasersin Medical Science, August 2007)

The PST method of the present invention, however, is based on theradiative processes (fluorescence, phosphorescence, luminescence, Raman,etc) whereas the PTT method is based on the radiationless processes (IC,YR and heat conversion) in molecules.

Non-Invasive Photonic Therapy

Current techniques such as photopheresis require removal and reinfusionof blood from patients, the proposed photonics based modalities canexcite non-invasively and directly from outside the body, FIG. 2illustrate the concept of non-invasive photonic therapy. The EEC canserve as an Energy Down-Converting (EDC) or Energy Up-Converting (EUC)material.

Photonics treatment modalities include both optical and non-opticaltechnologies that deal with electromagnetic radiation, which is theenergy propagated through space by electric and magnetic fields. Theelectromagnetic spectrum is the extent of that energy, ranging fromgamma rays and X rays throughout ultraviolet, visible, infrared,microwave and radio frequency energy.

Spectral Range of Light Used for PEPST

Theoretically the plasmonics enhanced effect can occur throughout theelectromagnetic region provided the suitable nanostructures, nanoscaledimensions, metal types are used. Therefore, the PEPST concept is validfor the entire electromagnetic spectrum, i.e, energy, ranging from gammarays and X rays throughout ultraviolet, visible, infrared, microwave andradio frequency energy. However, for practical reasons, visible and NIRlight are used for silver and gold nanoparticles, since the plasmonresonances for silver and gold occurs in the visible and NIR region,respectively. Especially for gold nanoparticles, the NIR region is veryappropriate for non-invasive therapy,

Photon Excitation in the Therapeutic Window of Tissue.

There are several methods using light to excite photoactivate compoundsnon-invasively. We can use light having wavelengths within the so-called“therapeutic window” (700-1300 nm). The ability of light to penetratetissues depends on absorption. Within the spectral range known as thetherapeutic window (or diagnostic window), most tissues are sufficientlyweak absorbers to permit significant penetration of light. This windowextends from 600 to 1300 nm, from the orange/red region of the visiblespectrum into 80) the NIR. At the short-wavelength end, the window isbound by the absorption of hemoglobin, in both its oxygenated anddeoxygenated forms. The absorption of oxygenated hemoglobin increasesapproximately two orders of magnitude as the wavelength shortens in theregion around 600 nm. At shorter wavelengths many more absorbingbiomolecules become important, including DNA and the amino acidstryptophan and tyrosine. At the infrared (IR) end of the window,penetration is limited by the absorption properties of water. Within thetherapeutic window, scattering is dominant over absorption, and so thepropagating light becomes diffuse, although not necessarily enteringinto the diffusion limit. FIG. 3 shows a diagram of the therapeuticwindow of tissue. The following section discusses the use of one-photonand multi-photon techniques for therapy.

Two methods that can be used for excitation: one-photon or multi-photonexcitation (MPE).

Multi-Photon Excitation for Energy Upconversion (UC)

One energy upconversion (UC) technique involves multi-photon excitation(MPE) discussed in a previous report. If the two-photon technique isused, one can excite the PA molecules with light at 700-1000 nm, whichcan penetrate deep inside tissue, in order to excite molecules thatabsorb in the 350-500 nm spectral region. This approach can excite thepsoralen compounds, which absorb in the 290-350 nm spectral region andemit in the visible. With one-photon method, the photo-activator (PA)drug molecules can directly absorb excitation light at 600-1300 nm. Inthis case we can design a psoralen-related system (e.g., psoralesnhaving additional aromatic rings) or use other PA systems: photodynamictherapy drugs, ALA, etc. In this MPE method the EEC material or the PAdrug is designed to absorb the multi-photon excitation energy and emit aphoton with energy higher than the original one-photon excitation.

Energy Upconversion Using Rare-Earth Doped Compound and SimilarMaterials

The process of energy upconversion using rare-earth doped materials havebeen extensively studied for photonics applications, its mostly knownarea of research being for UPC solid state lasers. See some refs on UCprocesses: (J. F. Suyver *, A. Aebischer, D. Biner, P. Gerner, J. Grimm,S. Heer, K. W. Krämer, C. Reinhard, H. U. Gü del Novel materials dopedwith trivalent lanthanides and transition metal ions showingnear-infrared to visible photon upconversion, Optical Materials 27(2005) 1111-1130); R. Paschotta, N. Moore, W. A. Clarkson, A. C.Trooper, D. C. Hanna, G. Maze, IEEE J Sel. Top. Quantum Electron. 3,1997.1100.; C. C. Ye, M Hempstead, D. W. Hewak, D. N. Payne, IEEEPhoton. Technol. Lett. 9_1997.1104; D. M. Baney, G. Ramkin, K. W Chang,Opt. Lett. 21_1996.1372; D. S. Funk, J. G. Eden, Proc. SPIE2841_1996.42.; B. R. Reddy and P. Venkateswarlu, Infrared to visibleenergy upconversion in Er ³⁺-doped oxide glass, Appl. Phys. Lett. 64,1327 (1994); G. S. Maciel*, A. Biswas and P. N. Prasad,Infrared-to-visible Eu ³⁺ energy upconversion due to cooperative energytransfer from an Yb ³⁺ ion pair in a sol-gel processed multi-componentsilica glass, Optics Communications, Volume 178, Issues 1-3, 1 May 2000,Pages 65-69; and Kapoor, Highly efficient infrared-to-visible energyupconversion in Er3+:Y2O3, Optics letters [0146-9592] yr: 2000 vol: 25pg: 338).

This method requires the use of a material that is designed to absorbthe multi-photon excitation energy and emit a photon with energy higherthan the original excitation photon. In general the energy up-convertingprocess involve an absorber ion and an emitter ion in a crystal. Amulti-photon (e.g., 2-photon) excitation energy (e.g., NIR) excites theabsorber ion, which transfers this energy radiationlessly to the emitterion that emits a photon that has an energy higher than the excitationphoton energy. Note there is a slight difference with the MPEupconverting method which does not necessarily require a materials(designed to absorb the multi-photon excitation energy). Three-photonupconversion processes involving energy transfer from Yb3+ to Tb3+ havebeen reported to produce efficient UV emission in the glass ceramiccontaining Tb3+/Yb3+:CaF2 nanocrystals [L. Huang, T. Yamashita, R. Jose,Y. Arai, T Suzuki, and Y. Ohishi, Appl. Phys. Lett. 90, 131116_2007_].Chen et al reported a Tm3+/Yb3+:β-YF3 nanocrystals embedded glassceramic which yields intense UV upconversion luminescence through thefour- or five-photon processes [-Daqin Chen, Yuansheng Wang, a_ YunlongYu, and Ping Huang, Intense ultraviolet upconversion luminescence fromTm3+/Yb3+:β-YF3 nanocrystals embedded glass ceramic, Appl. Phys. Lett,91, 051920, 2007]. Chen et al reported ultraviolet upconversionfluorescence in rare-earth-ion-doped Y₂O₃ induced by infrared diodelaser excitation [G. Y. Chen, G. Somesfalean, Z. G. Zhang, Q. Sun, andF. P. Wang, Optics Letters, Vol. 32, Issue 1, pp. 87-89]

Concept for Plasmonics-Enhanced Upconversion (PE-UC)

Basic Principle of Plasmonics and Enhanced Electromagnetic Fields

Where as the photothermal properties of plasmonics metal nanoparticleshave been used, to our knowledge the spectroscopic absorption andemission of plasmonics-active nanoparticles in phototherapy have notbeen reported.

To differentiate our proposed treatment modality from photothermaltherapy techniques (some of which also use gold nanoparticles forlocalized heating of tumors), we refer our technique to as“plasmonics-enhanced energy upconversion (PE-UC). In PEPST, theplasmonics-enhanced spectroscopic properties (spectral absorption,emission, scattering) are the major factors involved in the treatment.

The PEPST principle is based on the enhancement mechanisms of theelectromagnetic field effect. There are two main sources ofelectromagnetic enhancement: (1) first, the laser electromagnetic fieldis enhanced due to the addition of a field caused by the polarization ofthe metal particle; (2) in addition to the enhancement of the excitationlaser field, there is also another enhancement due to the moleculeradiating an amplified emission (luminescence, Raman, etc.) field, whichfurther polarizes the metal particle, thereby acting as an antenna tofurther amplify the Raman/Luminescence signal.

Electromagnetic enhancements are divided into two main classes: a)enhancements that occur only in the presence of a radiation field, andb) enhancements that occur even without a radiation field. The firstclass of enhancements is further divided into several processes. Plasmaresonances on the substrate surfaces, also called surface plasmons,provide a major contribution to electromagnetic enhancement. Aneffective type of plasmonics-active substrate consists of nanostructuredmetal particles, protrusions, or rough surfaces of metallic materials.Incident light irradiating these surfaces excites conduction electronsin the metal, and induces excitation of surface plasmons leading toRaman/Luminescence enhancement. At the plasmon frequency, the metalnanoparticles (or nanostructured roughness) become polarized, resultingin large field-induced polarizations and thus large local fields on thesurface. These local fields increase the Luminescence/Raman emissionintensity, which is proportional to the square of the applied field atthe molecule. As a result, the effective electromagnetic fieldexperienced by the analyte molecule on theses surfaces is much largerthan the actual applied field. This field decreases as 1/r³ away fromthe surface. Therefore, in the electromagnetic models, theluminescence/Raman-active analyte molecule is not required to be incontact with the metallic surface but can be located anywhere within therange of the enhanced local field, which can polarize this molecule. Thedipole oscillating at the wavelength λ of Raman or luminescence can, inturn, polarize the metallic nanostructures and, if λ is in resonancewith the localized surface plasmons, the nanostructures can enhance theobserved emission light (Raman or luminescence). There are two mainsources of electromagnetic enhancement: (1) first, the laserelectromagnetic field is enhanced due to the addition of a field causedby the polarization of the metal particle; (2) in addition to theenhancement of the excitation laser field, there is also anotherenhancement due to the molecule radiating an amplifiedRaman/Luminescence field, which further polarizes the metal particle,thereby acting as an antenna to further amplify the Raman/Luminescencesignal. Plasmonics-active metal nanoparticles also exhibit stronglyenhanced visible and near-infrared light absorption, several orders ofmagnitude more intense compared to conventional laser phototherapyagents. The use of plasmonic nanoparticles as highly enhancedphotoabsorbing agents has thus introduced a much more selective andefficient phototherapy strategy. The tunability of the spectralproperties of the metal nanoparticles and the biotargeting abilities ofthe plasmonic nanostructures make the PEPST method promising.

The novel PEPST concept is based on several important mechanisms:

-   -   Increased absorption of the excitation light by the plasmonic        metal nanoparticles, resulting in enhanced photoactivation of        drug molecules    -   Increased absorption of the excitation light by the plasmonic        metal nanoparticles that can serve as more efficient EEC        systems, yielding more light for increase excitation PA        molecules    -   Increased absorption of the excitation light by the photoactive        drug system adsorbed on or near the plasmonic metal        nanoparticles    -   Increased light absorption of the EEC molecules adsorbed on or        near the metal nanoparticles    -   Amplified emission from the EEC molecules adsorbed on or near        the metal nanoparticles    -   Increased absorption of emission light emitted by the EEC by the        PA molecule

One of several phenomena that can enhance the efficiency of lightemitted (Raman or luminescence). from molecules adsorbed or near a metalnanostructures Raman scatter is the SERS effect. In 1984, the PI'slaboratory first reported the general applicability of SERS as ananalytical technique, and the possibility of SERS measurement for avariety of chemicals including several homocyclic and heterocyclicpolyaromatic compounds [T. Vo-Dinh, M. Y. K. Hiromoto, G. M Begun and RL. Moody, “Surface-enhanced Raman spectroscopy for trace organicanalysis,” Anal. Chem., vol. 56, 1667, 1984]. Extensive research hasbeen devoted to understanding and modeling the Raman enhancement in SERSsince the mid 1980's. FIG. 4, for example, shows the early work byKerker modeling electromagnetic field enhancements for spherical silvernanoparticles and metallic nanoshells around dielectric cores as farback as 1984 [M. M Kerker, Acc. Chem. Res., 17, 370 (1984)]. This figureshows the result of theoretical calculations of electromagneticenhancements for isolated spherical nanospheres and nanoshells atdifferent excitation wavelengths. The intensity of the normally weakRaman scattering process is increased by factors as large as 10¹³ or10¹⁵ for compounds adsorbed onto a SERS substrate, allowing forsingle-molecule detection. As a result of the electromagnetic fieldenhancements produced near nanostructured metal surfaces, nanoparticleshave found increased use as fluorescence and Raman nanoprobes.

The theoretical models indicate that it is possible to tune the size ofthe nanoparticles and the nanoshells to the excitation wavelength.Experimental evidence suggests that the origin of the 10⁶- to 10¹⁵-foldRaman enhancement primarily arises from two mechanisms: a) anelectromagnetic “lightning rod” effect occurring near metal surfacestructures associated with large local fields caused by electromagneticresonances, often referred to as “surface plasmons”; and b) a chemicaleffect associated with direct energy transfer between the molecule andthe metal surface.

According to classical electromagnetic theory, electromagnetic fieldscan be locally amplified when light is incident on metal nanostructures.These field enhancements can be quite large (typically 10⁶- to 10⁷-fold,but up to 10¹⁵-fold enhancement at “hot spots”). When a nanostructuredmetallic surface is irradiated by an electromagnetic field (e.g., alaser beam), electrons within the conduction band begin to oscillate ata frequency equal to that of the incident light. These oscillatingelectrons, called “surface plasmons,” produce a secondary electric fieldwhich adds to the incident field. If these oscillating electrons arespatially confined, as is the case for isolated metallic nanospheres orroughened metallic surfaces (nanostructures), there is a characteristicfrequency (the plasmon frequency) at which there is a resonant responseof the collective oscillations to the incident field. This conditionyields intense localized field enhancements that can interact withmolecules on or near the metal surface. In an effect analogous to a“lightning rod,” secondary fields are typically most concentrated atpoints of high curvature on the roughened metal surface.

Among various materials, luminescent nanoparticles have attractedincreasing technological and industrial interest. In the context of thepresent invention, nanoparticle refers to a particle having a size lessthan one micron. While the description of the invention describesspecific examples using nanoparticles, the present invention in manyembodiments is not limited to particles having a size less than onemicron. However, in many of the embodiments, the size range of having asize less than one micron, and especially less than 100 nm producesproperties of special interest such as for example emission lifetimeluminescence quenching, luminescent quantum efficiency; andconcentration quenching and such as for example diffusion, penetration,and dispersion into mediums where larger size particles would notmigrate.

U.S. Pat. No. 4,705,952 (the contents of which are hereby incorporatedherein by reference) describes an infrared-triggered phosphor thatstored energy in the form of visible light of a first wavelength andreleased energy in the form of visible light of a second wavelength whentriggered by infrared light. In some cases, U.S. Pat. No. 4,705,952describes that “the upconversion continues for as long as several daysbefore a new short recharge is required.” The phosphors in U.S. Pat. No.4,705,952 were compositions of alkaline earth metal sulfides, rare earthdopants, and fusible salts. The phosphors in U.S. Pat. No. 4,705,952were more specifically phosphors made from strontium sulfide, bariumsulfide and mixtures thereof; including a dopant from the rare earthseries and europium oxide, and mixtures thereof; and including a fusiblesalt of fluorides, chlorides, bromides, and iodides of lithium, sodium,potassium, cesium, magnesium, calcium, strontium, and barium, andmixtures thereof. The materials described in U.S. Pat. No. 4,705,952 areuseful in various embodiments of the invention.

The energy relations present in the upconverter in U.S. Pat. No.4,705,952 are shown in the energy diagram of FIG. 1, where energy statesE and T are introduced by two selected impurities. Excitation of thesestates by absorption of light having a minimum energy of E minus G willcause electrons to be raised to the band at energy state E. Whencharging illumination ceases, many of the excited electrons will fall toenergy state T and remain trapped there. The trapping phenomenon isillustrated at the left of FIG. 1. Later exposure to triggeringillumination of infrared light can supply E minus T energies, permittingthe infrared-triggered phosphor in excited state T to transition tolevel E, as shown at the right of FIG. 5. A photon is emitted duringthis transition process. The resulting light emission is characterizedby a wavelength associated with E minus G.

If the depth of the trap is several times higher than the thermalenergy, more than 99% of the electrons are in the electron-hole trap. Ifthe depth of the traps is about 1 eV, then in the dark, most of thetraps are filled, band E is almost empty and electron hole recombinationis negligible. In some cases, U.S. Pat. No. 4,705,952 describes that“the storage times become extremely long, on the order of years.” Thematerial is thus adapted to receive infrared photons and to emit higherenergy photons in a close to 1:1 relation. With storage times this long,these infrared-triggered phosphors can be used in various embodiments ofthe present invention as a viable mechanism for both medical andnon-medical applications where commercial IR lasers are used to activatephosphorescence in a medium, thereby internally in a medium or in apatient generating visible or ultraviolet light.

Considerable effort has gone into the synthesis of luminescentnanoparticles, and numerous investigations of the optical propertieshave been performed. The synthesis of oxide nanoparticles such as thosethat are based on the lanthanides have been achieved by a number ofprocesses including solid-gel (sol-gel) techniques, gas phasecondensation or colloidal chemical methods. While efforts to makeconcentrated colloidal solutions of highly uniform size luminescentnanoparticles have met with some technical difficulties, synthesis ofuseful amounts of some 5 nanometer sized lanthanide doped oxides havebeen achieved as shown in a paper by Bazzi et al entitled Synthesis andluminescent properties of sub 5-nm lanthanide oxide particles, in theJournal of Luminescence 102 (2003) pages 445-450, the entire contents ofwhich are incorporated herein by reference. Materials such as these andthe other materials discussed below are useful materials forupconversion although the prior art to date has not concentrated onparticular application of these materials for materials, chemical,medical, pharmaceutical, or industrial processing. Indeed, the work byBazzi et al concentrated on understanding the properties on lanthanideoxide nanoparticles with an emphasis on the microstructural propertiesand optical emission properties (i.e. concentrated on the fluorescenceand down conversion properties of these materials). Nevertheless, thematerials described by Bazzi et al are useful in various embodiments ofthe invention.

The present inventors have realized that such upconversion materials canbe used in various materials, chemical, medical, pharmaceutical, orindustrial processing. In one example of others to be described below, ananoparticle of a lanthanide doped oxide can be excited with nearinfrared laser light such as 980 nm and 808 nm to produce bothultraviolet, visible, and near infrared light depending on the dopanttrivalent rare earth ion(s) chosen, their concentration, and the hostlattice. The ultraviolet, visible, and/or near infrared light can thenbe used to drive photoactivatable reactions in the host mediumcontaining the lanthanide doped oxide.

Other work reported by Suyver et al in Upconversion spectroscopy andproperties of NaYF ₄ doped with Er³⁺, Tm³⁺ and or Yb³⁺, in Journal ofLuminescence 117 (2006) pages 1-12, the entire contents of which areincorporated herein by reference, recognizes in the NaYF₄ materialsystem upconversion properties. Yet, there is no discussion as to thequality or quantity of upconverted light to even suggest that the amountproduced could be useful for various materials, chemical, medical,pharmaceutical, or industrial processing. The materials described bySuyver et al are useful in various embodiments of the invention.

Reference will now be made in detail to the present preferred embodimentof the invention, an example of which is illustrated in the accompanyingdrawings, in which like reference characters refer to correspondingelements.

FIG. 6 is a schematic reproduced from Suyver et al showing a schematicenergy level diagram of upconversion excitation and visible emissionsschemes for Er³⁺, Tm³⁺ and or Yb³⁺ ions Full, dotted, dashed, and curlyarrows indicate respectively radiative, non-radiative energy transfer,cross relaxation and other relaxation processes.

The lanthanide doped oxides differ from more traditional multi-photon upconversion processes where the absorption of, for example, two photonsis needed in a simultaneous event to promote an electron from a valencestate directly into an upper level conduction band state whererelaxation across the band gap of the material produces fluorescence.Here, the co-doping produces states in the band gap of the NaYF₄ suchthat the Yb³⁺ ion has an energy state at ²F_(5/2) pumpable by a singlephoton event and from which other single photon absorption events canpopulate even higher states. Once in this exited state, transitions tohigher energy radiative states are possible, from which light emissionwill be at a higher energy than that of the incident light pumping the²F_(5/2) energy state. In other words, the energy state at ²F_(5/2) ofthe Yb³⁺ ion is the state that absorbs 980 nm light permitting apopulation build up serving as the basis for the transitions to thehigher energy states such as the ⁴F_(7/2) energy state. Here,transitions from the ⁴F_(7/2) energy state produce visible emissions.

Chen et al have described a four photon upconversion in Four-photonupconversion induced by infrared diode laser excitation inrare-earth-ion-doped Y ₂ O ₃ nanocrystals, Chemical Physics Letters, 448(2007) pp. 127-131 In that paper, emissions at 390 nm and at 409 nm wereassociated with a four-photon upconversion process in the Y₂O₃nanocrystals. FIG. 7 reproduced below from Chen et al shows a ladder ofstates by which an infrared light source can progressively pump untilthe ⁴D_(7/2) state is reached. From this upper state, transitionsdownward in energy occur until the ⁴G_(1/2) state is reached, where atransition downward in energy emits a 390 nm photon. The materialsdescribed by Chen et al are useful in various embodiments of theinvention.

U.S. Pat. No. 7,008,559 (the entire contents of which are incorporatedherein by reference) describes the upconversion performance of ZnS whereexcitation at 767 nm produces emission in the visible range. Thematerials described in U.S. Pat. No. 7,008,559 (including the ZnS aswell as Er³⁺ doped BaTiO₃ nanoparticles and Yb³⁺ doped CsMnCl₃) aresuitable in various embodiments of the invention.

Further materials specified for up conversion in the invention includeCdTe, CdSe, ZnO, CdS, Y₂O₃, MgS, CaS, SrS and BaS. Such up conversionmaterials may be any semiconductor and more specifically, but not by wayof limitation, sulfide, telluride, selenide, and oxide semiconductorsand their nanoparticles, such as Zn_(1-x)Mn_(x)S_(y),Zn_(1-x)Mn_(x)Se_(y), Zn_(1-x)Mn_(x)Te_(y), Cd_(1-x)MnS_(y),Cd_(1-x)Mn_(x)Se_(y), Cd_(1-x)Mn_(x)Te_(y), Pb_(1-x)Mn_(x)S_(y),Pb_(1-x)Mn_(x)Se_(y), Pb_(1-x)Mn_(x)Te_(y), Mg_(1-x)MnS_(y),Ca_(1-x)Mn_(x)S_(y), Ba_(1-x)Mn_(x)S_(y) and Sr_(1-x), etc. (wherein,0<x≤1, and 0<y≤1). Complex compounds of the above-describedsemiconductors are also contemplated for use in the invention—e.g.(M_(1-z)N_(z))_(1-x)Mn_(x)A_(1-y)B_(y) (M=Zn, Cd, Pb, Ca, Ba, Sr, Mg;N=Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S, Se, Te, O; B=S, Se, Te, O; 0<x≤1,0<y≤1, 0<z≤1). Two examples of such complex compounds areZn_(0.4)Cd_(0.4)Mn_(0.2)S and Zn_(0.9)Mn_(0.1)S_(0.8)Se_(0.2).Additional conversion materials include insulating and nonconductingmaterials such as BaF₂, BaFBr, and BaTiO₃, to name but a few exemplarycompounds. Transition and rare earth ion co-doped semiconductorssuitable for the invention include sulfide, telluride, selenide andoxide semiconductors and their nanoparticles, such as ZnS; Mn; Er; ZnSe;Mn, Er; MgS; Mn, Er; CaS; Mn, Er; ZnS; Mn, Yb; ZnSe; Mn, Yb; MgS; Mn,Yb; CaS; Mn, Yb etc., and their complex compounds:(M_(1-z)N_(z))_(1-x)(Mn_(q)R_(1-q))_(x)A_(1-y)B_(y) (M=Zn, Cd, Pb, Ca,Ba, Sr, Mg; N=Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S, Se, Te, O; B=S, . . .0<z≤1, o<q≤1).

Some nanoparticles such as ZnS:Tb³⁺, Er³⁺; ZnS:Tb³⁺; Y₂O₃:Tb³⁺;Y₂O₃:Tb³⁺, Er3⁺; ZnS:Mn²⁺; ZnS:Mn,Er³⁺ are known in the art to functionfor both downconversion luminescence and upconversion luminescence.

Because upconversion stimulates or produces emission at shorterwavelengths, there are applications directed to medicine where thelonger wavelength light is more capable than a shorter wavelength lightof penetrating deep into biological tissue. Accordingly, withupconverter materials pre-positioned inside for example a biologicaltissue or an aqueous solution, the longer wavelength light (such as froma commercial IR laser) can be used in one embodiment to image deep skintissue (with the upconverter materials emitting visible or NIR light fordetection), and/or the longer wavelength light in one embodiment can beused to excite the upconverters in the biological tissue and thereafterproduce shorter wavelength light (e.g., ultraviolet light) to drivephotochemical or pharmaceutical reactions in the body. Details of theseparticular applications will be discussed in more detail later.

FIG. 8A is a schematic of a depiction of an upconverter material (i.e.,a photoactive material) according to one embodiment of the invention.FIG. 8A shows a number of structural configurations for placement of adielectric core upconverter material (which is of a nanometer sizedscale) in proximity to a metal shell. Incident light at a wavelength λ₁interacts with the upconverting dielectric core. The interaction oflight λ₁ with the dielectric core produces a secondary emission at afrequency λ₂ which has a shorter wavelength than λ₁ and accordingly hasa higher energy than λ₁. While the exact physical mechanisms for theupconversion may depend on the particular upconversion material andprocess being used in a particular application, for the purposes fordiscussion and illustration, the following explanation is offered.

In the context of FIG. 8A, when a wavelength λ₁ interacts with adielectric material core, three separate processes are well understoodfor the upconversion process involving trivalent rare earth ions. Thesethree processes are:

-   -   1) excited state absorption whereby two photons are absorbed        sequentially by the same ion to excite and populate one or more        states;    -   2) energy transfer upconversion which is a transfer of        excitation from one ion to another already in an excited state;        and    -   3) a cooperative process of multiphotons where two nearby ions        in excited states are emitting collectively from a virtual        state.

Regardless of which one of these processes is occurring between thechosen ion(s) and the host lattice, the end result is a photon of energygreater than the excitation energy being emitted from the host latticefor the upconversion process.

Therefore, the particular ion being activated (whether it be a dopantion or a host ion of a lattice such as in the neodymium oxide) will bechosen based on the host material being processed, in order that thedopant ion or the host ion in the dielectric core provide ion stateswhich are pumpable by the NIR source to generate the resultant emissionλ₂. While many of these materials have been studied in the past in thebulk state, prior to the invention, the targeted use of these materialsin the noncrystalline and nanosize range for various materials,chemical, medical, pharmaceutical, or industrial processing have notbeen exploited, especially at the size of dielectric cores and with theapplication of metallic shells.

Hence, the invention in one embodiment provides a nanoscale upconversionsystem for producing a photostimulated reaction in a medium. The systemincludes a nanoparticle configured, upon exposure to a first wavelengthλ₁ of radiation, to generate a second wavelength λ₂ of radiation havinga higher energy than the first wavelength λ₁. The system includes ametallic shell encapsulating at least a fraction of the nanoparticle andincludes a receptor disposed in the medium in proximity to thenanoparticle. The receptor upon activation by the second wavelength λ₂generates directly or indirectly the photostimulated reaction. In oneembodiment of the invention, a physical characteristic of the metallicstructure is set to a value where a surface plasmon resonance in themetallic structure resonates at a frequency which provides spectraloverlap with at least one of the first wavelength λ₁ and the secondwavelength λ₂.

Within the context of the invention, the term “physical characteristic”of the metallic shell or core can relate to any characteristic of themetal itself or the shell or core dimensions or shape which affects thesurface plasmon resonance frequency. Such physical characteristics caninclude, but are not limited to, a conductivity, a radial dimension, achemical composition or a crystalline state of the metal shell or core.

In various embodiments, the metallic structures can be a metallic shellencapsulating at least a fraction of the nanoparticle in the metallicshell wherein a conductivity, a radial dimension, or a crystalline stateof the metallic shell sets the surface plasmon resonance in the metallicstructure to resonate at a frequency which provides spectral overlapwith either the first wavelength λ₁ or the second wavelength λ₂. Invarious embodiments, the metallic structures can be a multi-layermetallic shell encapsulating at least a fraction of the nanoparticle inthe metallic shell wherein a conductivity, a radial dimension, or acrystalline state of the metallic shell sets the surface plasmonresonance in the metallic structure to resonate at the first wavelengthλ₁ and the second wavelength λ₂. This capability permits radiation at λ₁and λ₂ to be amplified.

In various embodiments, the metallic structures can be a metallicparticle existing in one or more multiple structures. These multiplestructures can have a variety of shapes including for example sphere,spheroid, rod, cube, triangle, pyramid, pillar, crescent, tetrahedralshape, star or combination thereof disposed adjacent the nanoparticlewherein a conductivity, a dimension (e.g. a lateral dimension or athickness), or a crystalline state of the metallic structure sets thesurface plasmon resonance in the metallic particle or rod to resonate ata frequency which provides spectral overlap with either the firstwavelength λ₁ or the second wavelength λ₂. Such shapes are described inthe present figures and in the figures in U.S. Ser. No. 12/401,478 whichis incorporated by reference in its entirety. The shape choice canaffect the frequency of the surface plasmon resonance. It is known thatthe plasmon band is changed by the shape of nanoparticles (e.g., prolateand obloid spheroids). The paper “Spectral bounds on plasmon resonancesfor Ag and Au prolate and oblate nanospheroids,” in the Journal ofNanophotonics, Vol. 2, 029501 (26 Sep. 2008), the entire contents ofwhich are incorporated by reference, shows plasmon resonance shifts forshaping of Ag and plasmon resonance shifts for shaping of Au of prolateand obloid spheroids. In one embodiment of the invention, with anincreasing aspect ratio for a metallic structure of the invention, theprolate spheroid resonance is red shifted relative to a sphere with nolower limit (under the assumptions of a Drude dispersion model). On theother hand, the oblate resonances are “blue shifted” as the spheroidbecomes increasingly flat, but up to a limit.

In various embodiments, the metallic structures can be a metallicstructure disposed interior to the nanoparticle wherein a conductivityor a dimension (e.g. a lateral dimension or a thickness) of the metallicstructure sets the surface plasmon resonance in the metallic structureto resonate at a frequency which provides spectral overlap with eitherthe first wavelength λ₁ or the second wavelength λ₂. In variousembodiments, the metallic structures can be a metallic multi-layerstructure disposed interior to the nanoparticle wherein a conductivityor a dimension (e.g. a lateral dimension or a thickness) of the metallicstructure sets the surface plasmon resonance in the metallic structureto resonate at the first wavelength λ₁ and the second wavelength λ₂.This capability once again permits radiation at λ₁ and λ₄ to beamplified.

In another embodiment, the invention provides a nanoparticle structureincluding a sub 1000 nm dielectric core and a metallic structuredisposed in relation to the nanoparticle. The dielectric core includesat least one of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄, YAG, YAP, Nd₂O₃, LaF₃,LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, Na-doped YbF₃, or SiO₂. Suchnanoparticle structures can exhibit in certain embodiments surfaceplasmon resonance in the metallic structures to enhance upconversion oflight from a first wavelength λ₁ to a second wavelength λ₂.

In another embodiment, the invention provides a nanoparticle structureincluding a sub 10 nm dielectric core and a metallic shell encapsulatingat least a fraction of the nanoparticle. The dielectric core includesany of the dielectric cores noted above. Such nanoparticle structurescan exhibit in certain embodiments surface plasmon resonance in themetallic shell to enhance upconversion of light from a first wavelengthλ₁ to a second wavelength λ₂.

As described above, a metallic structure is in particular designed witha layer thickness to enhance the photon upconversion process throughplasmonic enhancement. The thickness of the structure is “tuned” in itsthickness to the absorption process by having a dimension in whichplasmons (i.e., electrons oscillations) in the structure have aresonance in frequency which provides spectral overlap with theabsorption band targeted. Thus, if the upconversion is to be stimulatedby 980 nm NIR light, then the thickness of the structure is “tuned” in athickness to where a plasmon resonance resonates at a frequency also of980 nm (or in the neighborhood thereof as plasmon resonances aretypically broad at these wavelengths).

Such a plasmon resonating structure can be made of numerous transitionmetals, including though not limited to gold, silver, platinum,palladium, nickel, ruthenium, rhenium, copper, and cobalt. When formedof a gold nanoshell, the recommended thickness to resonate with 980 nmlight is approximately 3.5 nm surrounding an 80 nm upconverting core, asprojected by extended Mie theory calculations. (See Jain et at,Nanolett. 2007, 7(9), 2854 the entire contents of which are incorporatedherein by reference.) FIG. 8B is reproduced from Jain et al andillustrates the capability in the present invention to “tune” the metalshell to have a spectral overlap with the excitation and/or emissionradiation wavelengths. This capability of matching or tuning of thefrequencies provides an enhancement of the absorption which would not bepresent with a dielectric core alone.

In one embodiment of the invention, the metallic structures can be analloy such as for example a Au:Ag alloy. The alloy content can be set toadjust the frequency of the surface plasmon resonance. For instance, thealloy content may be one factor providing a surface plasmon resonance at365 nm. In one embodiment, specifically a silver concentration of 65 to75%, and more specifically a silver concentration of 67% is used for a365 nm surface plasmon resonance. In: one embodiment of the invention,the metallic structures can be an alloy such as for example a Pt:Agalloy. The alloy content can be set to adjust the frequency of thesurface plasmon resonance. In one embodiment of the invention, themetallic structures can be an alloy such as for example a Pt:Au alloy.The alloy content can be set to adjust the frequency of the surfaceplasmon resonance.

In one embodiment of the invention, the nanoparticle can be an alloy oftwo or more materials. In this embodiment, the alloy can have acomposition between the two or more materials which is set to acompositional value where excitation of the alloy at first wavelength λ₁produces emission at the second wavelength λ₂. In one embodiment of theinvention, the nanoparticle can be a zinc sulfide and zinc selenidealloy. In one embodiment of the invention, the nanoparticle can be azinc sulfide and cadmium sulfide alloy.

In one embodiment of the invention, the zinc sulfide and zinc selenidenanoparticle alloy can have an alloy content set to provide a surfaceplasmon resonance at 365 nm and specifically having a zinc sulfideconcentration of 65 to 75%, and more specifically a zinc sulfideconcentration of 67%. In one embodiment of the invention, the zincsulfide and cadmium sulfide nanoparticle alloy can have an alloy contentis set to provide a surface plasmon resonance at 365 nm and specificallyhaving a zinc sulfide concentration of 65 to 75%, and more specificallya zinc sulfide concentration of 67%.

Some techniques for producing nanoparticles and nanoparticle alloyswhich are suitable for the invention are described in the followingdocuments, all of which are incorporated herein in their entirety: U.S.Pat. Nos. 7,645,318; 7,615,169; 7,468,146; 7,501,092; U.S. Pat. Appl.Publ. No. 2009/0315446; 2008/0277270; 2008/0277267; 2008/0277268; and WO2009/133138.

In one embodiment of the invention, the nanoparticle can be a dielectricor semiconductor configured to generate the wavelength λ. In oneembodiment of the invention, the nanoparticle can include multipledielectrics or semiconductors respectively configured to emit atdifferent wavelengths for. In one embodiment of the invention, multiplenanoparticles having different dielectrics or semiconductors can beincluded in a mixture of the nanoparticles dispersed in the medium.

In one embodiment of the invention, a quantum dot mixture can be usedfor the multiple nanoparticles. Quantum dots are in general nanometersize particles whose energy states in the material of the quantum dotare dependent on the size of the quantum dot. For example, quantum dotsare known to be semiconductors whose conducting characteristics areclosely related to the size and shape of the individual crystal.Generally, the smaller the size of the crystal, the larger the band gap,the greater the difference in energy between the highest valence bandand the lowest conduction band becomes. Therefore, more energy is neededto excite the dot, and concurrently, more energy is released when thecrystal returns to its resting state. In fluorescent dye applications,this equates to higher frequencies of light emitted after excitation ofthe dot as the crystal size grows smaller, resulting in a color shiftfrom red to blue in the light emitted.

Specifically, in one embodiment of the invention, a quantum dot mixture(QDM) coating can be deposited using CVD and or sol-gel techniques usingstandard precipitation techniques. The QDM coating can be made of asilicate structure that does not diminish UV output. Within the silicatefamily, silica (SiO₂) is suitable since it maximizes UV transmissionthrough the coating. The coating can further include a second layer of abiocompatible glass. Such bio-compatible glass and glass ceramiccompositions can contain calcium, a lanthanide or yttrium, silicon,phosphorus and oxygen. Other biocompatible materials and techniques aredescribed in the following patents which are incorporated herein intheir entirety: U.S. Pat. Nos. 5,034,353; 4,786,617; 3,981,736;3,922,155; 4,120,730; and U.S. Pat. Appl. Nos. 2008/0057096;2006/0275368; and 2010/0023101.

Since the present invention is directed towards use of these variousstructures within a living patient, when the exposed portion of theparticle comprises a potentially cytotoxic compound, it may be necessaryto add a further coating or shell of a biocompatible and biostablesubstance, such as SiO2, in order to avoid toxicity within the patient.

In one embodiment of the invention, the thickness of the metal shell isset depending on the absorption frequency (or in some cases the emissionfrequency) of the particular dopant ions in the dielectric core toenhance the total efficiency of the emission process of the upconvertedlight. Accordingly, the thickness of the metal shell can be consideredas a tool that in one instance enhances the absorption of λ₁, and inanother instance can be considered as a tool that enhances the emissionof λ₂, or in other situations can be considered an enhancement featurethat in combination enhances the overall net process.

Additionally, plasmon-phonon coupling may be used to reduce a resonancefrequency through the tuning of the bands to a degree off resonance.This may be useful in optimizing resonance energy transfer processes forthe purpose of coupling the core-shell nanoparticles to sensitivechromophores or drug targets. Accordingly, when a recipient 4 is outsideof the shell, the recipient 4 will receive enhanced light λ₂ by theabove-described plasmonic effect than would occur if the shell wereabsent from the structure.

In one example, FIG. 8A-1 shows UV-visible absorption spectra of cubicY2O3 (lower trace) and gold-coated Y2O3 (upper trace) dispersed using 10mM tri-arginine. Details of the preparation of the nanoparticle systemare provided below. The absorption spectrum of Y2O3 alone (lower trace)is fairly featureless, showing absorption due to the tri-arginine near200 nm and a gentle slope associated with scattering and absorption bythe Y2O3 nanoparticles extending into the visible portion of thespectrum. The gold-coated Y2O3 (upper trace), on the other hand, exhibita strong absorption band at 546 nm, which is characteristic of theplasmonics resonance band due to the gold shell around the Y2O3 cores.This feature is a plasmon band. If this feature were due to solid goldnanoparticles in solution, this feature would be centered at or below530 nm. Moreover, red-shifting of the plasmon absorption to 546 nm isconsistent with the presence of a gold shell around a dielectric core.

In one embodiment of the invention, the materials for the upconverterdielectric core can include a wide variety of dielectric materials, asdescribed above. In various embodiments of the invention, theupconverter dielectric core includes more specifically lanthanide dopedoxide materials. Lanthanides include lanthanum (La), cerium (Ce),praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm),europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium(Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu).Other suitable dielectric core materials include non-lanthanide elementssuch as yttrium (Y) and scandium (Sc). Hence suitable dielectric corematerials include Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄, Na-doped YbF₃, YAG, YAP,Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, or SiO₂. Thesedielectric cores can be doped with Er, Eu, Yb, Tm, Nd, Tb, Ce, Y, U, Pr,La, Gd and other rare-earth species or a combination thereof.

Lanthanides usually exist as trivalent cations, in which case theirelectronic configuration is (Xe) 4f^(n), with n varying from 1 (Ce³⁺) to14 (Lu³⁺). The transitions within the f-manifold are responsible formany of the photo-physical properties of the lanthanide ions, such aslong-lived luminescence and sharp absorption and emission lines. Thef-electrons are shielded from external perturbations by filled 5s and 5porbitals, thus giving rise to line-like spectra. The f-f electronictransitions are LaPorte forbidden, leading to long excited statelifetimes, in the micro- to millisecond range.

Accordingly, examples of doped materials in the invention include oxidessuch as yttrium oxide and neodymium oxide and aluminum oxide as well assodium yttrium fluoride and nanocrystalline perovskites and garnets suchas yttrium aluminum garnet (YAG) and yttrium aluminum perovskite (YAP).Of these materials, doping is required for some, but not all of thesematerials, for promoting upconversion efficiencies. In variousembodiments of the invention, the host nanocrystals are doped withtrivalent rare earth lanthanide ions from those lanthanide serieselements given above.

More specifically, in various embodiments of the invention, pairs ofthese dopants are introduced in order to make accessible more energystates in the host crystal. The activation and pumping of these energystates follows closely the principles discussed above with regard toFIG. 7. Doping concentrations in the invention can range from 0.2% to20% roughly per ion into the host lattice or in a weight or mol %variation. The efficiency of the upconversion processes of specificbands in these materials can be modulated by the percentages doped toinduce and enhance targeted emissions. Lanthanide doped upconverterswhile not limited to, can use the following mol percent dopantcompositions: 5% Er, 10% Yb, 0.2% Tm+3% Yb, and 1% Er+10% Yb.

The size of the nanocrystal will also have an effect on the efficiencyof the upconversion process, as a larger nanocrystal will have moresites for dopant ions to be accommodated into the host lattice,therefore enabling more emissions from the same doped host than if thenanocrystal were smaller. While the dopant percentages listed above arenot rigidly fixed, these numbers provide rudimentary teachings of thetypical percentages one would use in obtaining a particular dielectriccore material of the invention.

Moreover, some of these host crystals (e.g., neodymium oxide) in oneembodiment of the invention may require no specific doping to facilitateupconversion, which has been seen in one instance in Nd₂O₃ with anexcitation wavelength of 587 nm producing emissions at 372 nm, 402 nm,and 468 nm. See Que, W et al. Journal of Applied. Physics 2001, vol 90,pg 4865, the entire contents of which are incorporated herein byreference, Doping neodymium oxide with Yb³⁺, in one embodiment of theinvention, would enhance upconversion through sensitizing the Nd³⁺ ionswith a lower energy Yb³⁺ activator.

In one embodiment of the invention, the dielectric core is coated, suchas for example with a metallic shell 4, to enhance electron-phononcoupling and thereby increase upconversion efficiency, as discussedabove. In another embodiment of the invention, the shell can include aSiO₂- and/or TiO₂-coating, and this coating is in one embodiment coatedon doped Y₂O₃ upconverting nanoparticles to thereby, in some instances,increase the upconversion efficiency relative to an uncoatednanocrystal. Further, in one embodiment of the invention, the coatingcan be a polymer. In one embodiment, this coating is provided onNaYF₄:Ln/NaYF₄ dielectric core. Such coatings can increase theupconversion efficiency relative to an uncoated upconverter,

In another embodiment of the invention, phonon modes of an undopedhost-lattice (e.g., Y₂O₃) nanocrystals are modulated, for example, byAu, Ag, Pt, and Pd shells 4 of varying thicknesses. In variousembodiments of the invention, the upconverter dielectric core and theshell 4 system includes as upconverting nanocrystals Y₂O₃:Ln with NaYF₄shells, Y₂O₃:Ln with Au(Ag,Pt) shells, NaYF₄:Ln with Y₂O₃ shells,NaYF₄:Ln with Au(Ag,Pt) shells. In this system, the core diameter andshell outer/inner diameter of the metallic coatings can be set todimensions that are expected to be tunable to a plasmon mode overlap.

In other embodiments as discussed below, the metal coating or themetallic structure can exist inside the dielectric and the relativeposition of the metal structure to the dielectric structure can enhanceplasmon resonance. These structures with the metallic structure insidecan be referred to as a metallic core up converter or a metallic coredown converter. The metallic core technique for energy conversion isuseful since it takes advantage of metal nano-particles that haveimproved surface morphology compared to shell coatings on coredielectrics. The metal or metallic alloy in the inner core metallicenergy converter can be selected to tune its plasmonic activity. Thesestructures with the metallic structure outside can be referred to as acore up converter or a core down converter. These core up converter or acore down converter structures offer advantages for biocompatibility asthe core materials can be surrounded in a gold biocompatible shell.

FIG. 8C is a schematic illustration of a process for forming and aresultant Ln-doped Y₂O₃ core with a Au shell. One illustrative methodfor producing sub-10 nm Ln-doped Y₂O₃ nanoparticles with a metal shellcan be achieved through the polyol method. See Bazzi, R. et al. Journalof Luminescence, 2003, 102-103, 445-450, the entire contents of whichare incorporated by reference. In this approach, yttrium chloridehexahydrate and lanthanum-series chloride hexahydrates are combined inan appropriate ratio with respect to their cation concentration intosuspension with diethylene glycol (0.2 mol chloride per liter of DEG).To this suspension is added a solution of NaOH and water (0.2 mol/L and2 mol/L, respectively). The suspension is heated to 140° C. in a solventrecondensing/reflux apparatus for a period of 1 hour. Upon completion ofthe 1 hour of heating the solution has become transparent and nucleationof the desired nanoparticles has occurred. The temperature is thenincreased to 180° C. and the solution is boiled/refluxed for 4 hoursyielding Y₂O₃:Ln nanoparticles. This solution is then dialyzed againstwater to precipitate the nanoparticles or solvent is distilled off andexcess water added to precipitate the same. The nanoparticles arecollected through centrifugation and dried in vacuo.

The dried nanoparticles are then calcined at 900° C. for 2 hours toafford single phase, cubic Y₂O₃ nanocrystals with lanthanide dopantsequally distributed through the Y₂O₃ nanocrystal. This methodology maybe modified to allow for synthesis in a pressurized environment, therebyallowing for complete expression in the cubic phase, allowing for ashorter calcining times and lower temperatures leading to lessnanoparticle agglomeration and size growth.

Nanocrystals are then resuspended in toluene with sonication and treatedwith 2-triethoxysilyl-1-ethyl thioacetate (300 mM) in toluene. Volatilecomponents of the reaction mixture are removed in vacuo and theremaining residue is resuspended in toluene and treated with NaSMe.Volatile components of the reaction mixture are again removed in vacuoand the remaining residue is purified through reprecipitation,centrifugation, and drying. The thiol-terminated, surface-modifiednanocrystals are then resuspended in 0.1 M DDAB (didodecylammoniumbromide) in toluene and a solution of colloidal gold nanoparticles (˜1nm in diameter) coated in dodecylamine (prepared as per Jana, et al. J.Am. Chem. Soc. 2003, 125, 14280-14281, the entire contents of which areincorporated herein by reference) is added. The gold shell is thencompleted and grown to the appropriate shell thickness through additionsof AuCl₃ and dodecylamine in the presence of reducing equivalents oftetrabutylammonium borohydride. Thiol terminated organic acids may thenbe added to allow for increased water solubility and the completed goldmetal shell, Ln-doped, Y₂O₃ nanoparticles may be separated in thepresence of water through extraction or dialysis.

FIG. 8D is a schematic illustration of a process for forming and aresultant Ln-doped Y₂O₃ core with a NaYF₄ shell. In this embodiment ofthe present invention, Ln-doped Y₂O₃ cores for example may be shelledwith NaYF₄, Nd₂O₃, Ga₂O₃, LaF₃, undoped Y₂O₃, or other low phonon modedielectric material using a secondary polyol approach following silylprotection of the core nanocrystal. It has been shown that low phononmode host lattices (such as Y₂O₃, NaYF₄, etc.) are useful for aiding inthe upconversion process. This has been attributed to the nature ofelectron-phonon coupling to low phonon modes and the removal ofnon-radiative decay processes within the host-lattice/ion crystal.Accordingly, in one embodiment of the present invention, the dielectriccore materials are made of low mode phonon host lattices (such as Y₂O₃,Y₂O₂S, NaYF₄, NaYbF₄, YAG, YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄,YVO₄, YbF₃, YF₃, Na-doped YbF₃, or SiO₂, or alloys or combinationsthereof, etc.).

Different sized Y₂O₃ NPs can also be synthesized via a combustion methoddeveloped by Song et al. In this method, Y(NO₃)₃ and glycine solutionwere heated to evaporate excess water until spontaneous ignitionoccurred. Cubic Y₂O₃ NPs can be obtained upon 2 hr of annealing at 500°C. One advantage of this method is that the Y₂O₃ particle size can bechanged by varying the ratio between Y(NO₃)₃ and glycine. Anotheradvantage is that different ratios of dopants (e.g. Yb and Er) can beadded in the Y₂O₃ precursor solution and different doped Y₂O₃ NPs whichhave different emission properties can thus be synthesized. Due toinsolubility, Y₂O₃ NPs are known to form precipitate in water. Uponfunctionalization with glutamic acid, Y₂O₃ NPs can result in a goodsuspension in water and the well-dispersed NPs shown in FIGS. 9-1-A and9-1-B. XRD measurement showed that the as-synthesized Y₂O₃ NPs have acubic structure and this crystal structure is further proved by thelattice spacing as shown in FIGS. 9-1-A and 9-1-B. Ligand substitutionwith excess 3-mercaptopropionic acid or 3-mercaptopropylphosphonic acidin the presence of refluxing diethylene glycol can then be used tofunctionalize these particles with Au nanoparticles in similar fashionto treatment with mercaptoalkylsilanes, as is described below.

Nanocrystals are then resuspended in toluene with sonication and treatedwith 2-triethoxysilyl-1-ethyl thioacetate (300 mM) in toluene. Volatilecomponents of the reaction mixture are removed in vacuo and theremaining residue is resuspended in toluene and treated with NaSMe.Volatile components of the reaction mixture are again removed in vacuoand the remaining residue is purified through reprecipitation,centrifugation, and drying. The thiol-terminated, surface-modifiednanocrystals are then resuspended in 0.1 M DDAB (didodecylammoniumbromide) in toluene and a solution of colloidal gold nanoparticles (˜1nm in diameter) coated in dodecylamine (prepared as per Jana, et al. J.Am. Chem. Soc. 2003, 125, 14280-14281, the entire contents of which areincorporated herein by reference) is added. The gold shell is thencompleted and grown to the appropriate shell thickness through additionsof AuCl₃ and dodecylamine in the presence of reducing equivalents oftetrabutylammonium borohydride. Thiol terminated organic acids may thenbe added to allow for increased water solubility and the completed goldmetal shell, Ln-doped, Y₂O₃ nanoparticles may be separated in thepresence of water through extraction or dialysis.

Dried Y₂O₃ nanoparticles are resuspended in toluene with sonication andtreated with 2-triethoxysilyl-1-propionic acid, benzyl ester (300 mM) intoluene. Volatile components of the reaction mixture are removed invacuo and the remaining residue is resuspended in toluene and treatedwith a strong base. Volatile components of the reaction mixture areagain removed in vacuo and the remaining residue is purified throughreprecipitation, centrifugation, and drying. The carboxyl-terminated,surface-modified nanocrystals are then resuspended in a solution ofsodium fluoride in DEG and treated with yttrium nitrate hexahydrate atroom temperature, stirring for 12 hours (for NaYF₄ exemplar). Thereaction mixture is then brought to 180° C. for 2 hours to grow theNaYF₄ shell through Ostwald ripening. Nanoparticles are purified throughreprecipitation, as described previously. Organic acid terminatedpolymers, polyethylene glycol, polyethynyleneimine, or other FDAapproved, bioavailable polymer may then be added to allow for increasedwater solubility and the completed NaYF₄ shell, Ln-doped, Y₂O₃nanoparticles may be resuspended in water for medical use.

In various embodiments of the invention, the upconverter dielectric corecan be coated with thiol-terminated silanes to provide a coating of SiO₂about the core of similar reactivity to Y₂O₃. These thiolatednanoparticles are then exposed to colloidal Au (1-2 nm) which associatesto the nanoparticle surface and, with addition of HAuCl₄ and a reducingagent, Ostwald ripening coalesces the Au surface into a uniform shell ofa designated thickness. Solubility enhancement of NaYF₄ and other CaF₂lattices can be increased by the use of coupled trioctylphosphine-oleicamine, polyethylene glycol, and polyethyleneimine surfactants. Thesesurfactants associate to the surface of the nanoparticles withfunctional head groups and are soluble in either organic or aqueoussolvents to permit colloidal suspension of the nanoparticles

In one embodiment of the invention, the above-described methodology isused to synthesize novel upconverting core-shell nanoparticles ofY₂O₃:Ln with NaYF₄ shells, Y₂O₃:Ln with Au(Ag,Pt) shells, NaYF₄:Ln withY₂O₃ shells, NaYF₄:Ln with Au(Ag,Pt) shells where core and shelldiameters varying from 2 to 20 nm. In these novel material systems, thetuned ratio of core-to-shell diameter may permit a plasmon-phononresonance which should amplify absorption of NIR light and/orupconverted emission. In these novel material systems, control of thecore and shell diameters is one factor determining the size dependenteffect and subsequent tuning of plasmon-phonon resonance.

In one embodiment of the invention, this methodology is used tosynthesize novel mixed core-shell materials can include semiconductingY₂O₃ and NaYF₄ cores doped with various Ln series metals, which havebeen shown to possess large upconverting efficiencies. These doped Y₂O₃and NaYF₄ cores will have shells of Au(Ag,Pt, Pd) or undoped Y₂O₃ andNaYF₄ matrices which have the potential to enhance or tune the phononmodes needed for energy transfer in the upconversion process. Solubilitycan be enhanced, for example, by addition of thiolated organics (Aushell), organic chain triethanolsilane (Y₂O₃ shell), andtrioctylphospine-oleic amine (NaYF₄ shell). All core-shell nanoparticlesmay further be solublized into a colloidal suspension with the additionof triarginine peptide, polyethylene glycol, and polyethyleneiminesurfactants.

Since Y₂O₃ nanocrystals have a scintillation emission (down conversion)optimal for exciting drug derivatives of potential clinical importance,smaller nanocrystals offer advantages for biological targetingapplications. Given the permeability of biological tissues to X-rayirradiation, down conversion from X-rays to visible light through Y₂O₃nanocrystals offers a means of detecting the presence of nanoparticlescoupled to biological malignancies (e.g. cancer, autoimmune degeneratedtissue, foreign contaminants) through antibodies, Fab fragments, orcell-surface receptor specific peptides linked to the nanoparticlesurface. Subsequently, down converting nanoparticles offer a means ofgenerating UV/VIS/NIR light for photoactive drug activation directly atthe treatment site, deep within biological tissue where UV and VIS light(if applied externally) would likely not penetrate. Furthermore,upconverting Y₂O₃:Ln nanocrystals can be utilized in one embodiment ofthe invention for their absorption and emissive properties within theNIR window applicable for medical imaging.

In one embodiment of the invention, small nanocrystals of thesematerials are prepared using rare-earth (RE) precursors (e.g. chloride,nitrate, alkoxides) which are mixed with a defined amount of water in ahigh boiling polyalcohol (e.g., diethylene glycol) solvent. Thedehydrating properties of the alcohol and the high temperature of thesolution promote a non-aqueous environment for the formation of oxideparticles, as opposed to hydroxide, particles. Other solvents which canbe used include: ethylene glycol, triethylene glycol, propylene glycol,dipropylene glycol, tripropylene glycol, etc. (thereby providingsolvents with different boiling points). With these procedures, oneexpects sub-5 nm nanocrystals to be coated with Au, Ag, Pt, Pd (orcombinations thereof) layers. FIG. 9 illustrates one such coated sub-5nm nanocrystal.

Accordingly the synthesis of these nanocrystals and other dielectriccore elements can follow the methods described below.

In particular, one method of forming yttrium oxide nanocrystalsaccording to the present invention is to obtain precursors of theyttrium and rare earth ions in their salt forms, preferably in achloride salt of the hexahydrate form, which is more soluble thannon-hexahydrate forms. These salts are then combined in the correctmolar ratios as listed below to create a yttrium oxide containingsolution in a high boiling polyalcohol solvent with an added base of thecorrect proportion. An initial cation concentration of 0.2 moles perliter is mixed with a sodium hydroxide solution in water (0.2 moles perliter of sodium hydroxide per liter of reaction solution; 2 moles of H₂Oper liter per solution). The precursors were added together in thepolyalcohol solvent, stirred for one hour at 140° C. After the salts arecompletely dissolved, the solution is brought to reflux at 180° C. andheated for four hours. The reaction is then cooled to room temperatureyielding a transparent colloidal suspension of rare earth doped, yttriumoxide nanocrystals. The purification of this colloid produces the basicnanometer size of dielectric core shown in FIG. 9. The metallic shellcan then be prepared using the processes described below.

Similar methods can be employed for the preparation of the otherupconversion materials described above, such as for example for thepreparation of

1) nanoparticles of 2% neodymium and 8% ytterbium doped yttrium oxide,

2) europium and ytterbium doped yttrium oxide, and

3) any combination of rare earth trivalent ions doped into a neodymiumoxide nanocrystal.

In another embodiment of the invention, NaYF₄ dielectric particles havebeen fabricated with individual particles in the ˜70-200 nm size rangeas shown in FIG. 9-2. To produce these particles NaCl, TmCl₃, YCl₃ andYbCl₃ stock solutions (0.2M) were prepared by dissolving thecorresponding chlorides in water. A PEI stock solution (5%) was preparedby dissolving PEI (M_(n)˜10,000) in water. 10 mL NaCl solution, 8 mLYCl₃ solution, 1.8 mL YbCl₃ solution and 0.2 mL TmCl₃ solution wereadded to a round-bottom flask containing 60 mL of ethanol and 20 mL ofPE solution. After stirring at room temperature for approximately 10minutes, 2 mmol of NH₄F was added and the solution was stirred for anadditional 10 minutes. The solution was then transferred to aTeflon-lined autoclave which was placed in an oven at 200° C. for 24hours. After cooling to room temperature, the particles were isolated bycentrifugation and then washed three times using 50/50 H₂O-ethanol. Awhite powder was obtained after rotary evaporation.

Further, NaYF₄ dielectric particles have been produced and isolated intodispersed particles with two size distributions of ˜50 nm and ˜150 nm,as shown in FIG. 9-3. The procedure to generate these particles is thesame as that listed above, except that the YbCl₃ stock solution wasprepared by dissolving Yb₂O₃ in HCl. Further, YbF₃ dielectric particleshave been produced and isolated into homogeneous particles of a size of35 nm+/−5 nm, as shown in FIG. 9-4. Generation of these particles wassimilar to that listed above, except that the concentrations of all thesalts were halved (the PEI concentration remaining constant), and YbCl₃was used instead of YCl₃. As such, two YbCl₃ stock solutions (0.1 M)were prepared; the first by dissolving YbCl₃.6H₂O in water and thesecond by dissolving Yb₂O₃ in concentrated hydrochloric acid. Theremainder of the synthetic methodology remained the same. An opticalemission spectrum from these NaYF₄ dielectric core particles, excited at980 nm, is shown in FIG. 9-5.

In another embodiment of the invention, NaYbF₄ dielectric particles havebeen fabricated with individual particles in the ˜20-200 nm size rangeas shown in FIGS. 9-6, 9-7, 9-8, and 9-9. These particles were generatedthrough a thermal decomposition method based on the work of Boyer, J-C.et al. Nano Lett., 2007, 7(3), 847-852 and Shan, J. et al.Nanotechnology, 2009, 20, 275603-275616, the entire contents of whichare incorporated by reference. The particles were prepared by composinga slurry of NaTFA (2.5-4 mmol), 34 mL 1-octadecene, and 6 mL oleic acid,Y(TFA)₃, Yb(TFA)₃, and Ln(TFA)₃ (Ln=Tm) in given proportion totaling 2mmol of trifluoroacetate salt. The slurry was heated under vigorousstirring to 125° C. in a 100 mL, 2-neck round bottom flask with magneticstir bar and reflux condenser until full dissolution occurred and anyresidual water was removed through a vent needle. 6 mL trioctylphosphineor oleic acid was then added. The reaction apparatus was thentransferred to a molten salt bath (KNO₃:NaNO₃; 50:50 by mol %) held attemperatures varying from 350-414° C. and held at temperature for 15-60minutes. The reaction was then cooled to RT, poured into an equivalentvolume of absolute ethanol, sonicated, vortexed, and centrifuged at 21krcf (approx. 14k RPM) for 30 minutes. The resulting pellet wasresuspended and centrifuged in similar fashion with hexanes, followed bytwo washes of 50:50; water:ethanol, and a final wash of absoluteethanol. The purified nanocrystals were then dried in air overnight.

FIG. 10 shows some of the various embodiments of the upconverterstructures of the invention that can be designed: (a) a structureincluding upconverter (UC) molecules bound to a metal (gold)nanoparticle; (b) a structure including an UC-containing nanoparticlecovered with metal nanoparticles, (c) a metal nanoparticle covered withan UC-containing nanocap; (d) an UC-containing nanoparticle covered withmetal nanocap, (e) a metal nanoparticle covered with UC nanoshell, (f)an UC-containing nanoparticle covered with metal nanoshell, (g) anUC-containing nanoparticle covered with metal nanoshell with protectivecoating layer. The configurations (while shown in the FIG. 10 serieswith UC-containing materials) would be applicable for enhancement fordown converting materials. Moreover, in one embodiment of the invention,dielectric spacers (for examples silicates as discussed below) can beused with the structure of FIG. 10A-b to space apart the particle typemetallic structures. In another embodiment of the invention, dielectricspacers can be used with the structure of FIG. 106A-d, f to space apartthe metal layers, whether or not these layers are partial metal layersas in FIG. 10A-d or continuous metal layers as in FIG. 10A-f. See FIGS.10D-b, d, and f.

The plasmonic properties of various metallic structures, which have beeninvestigated in the art and are suitable for the invention, includemetallic nanoshells of spheroidal shapes [S. J. Norton and T. Vo-Dinh,“Plasmonic Resonance of Nanoshells of Spheroidal Shape”, IEEE Trans.Nanotechnology, 6, 627-638 (2007)], oblate metal nanospheres [S. J.Norton, T. Vo-Dinh, “Spectral bounds on plasmon resonances for Ag and Auprolate and oblate nanospheroids”, J. Nanophotonics, 2, 029501 (2008)],linear chains of metal nanospheres [S. J. Norton and T. Vo-Dinh,“Optical response of linear chains of metal nanospheres andnanospherolds”, J. Opt. Soc. Amer., 25, 2767 (2008)], gold nanostars [C.G. Khoury and T. Vo-Dinh, “Gold Nanostars for Surface-Enhanced RamanScattering: Synthesis, Characterization and Applications”, J. Phys. ChemC. 112, 18849-18859 (2008)], nanoshell dimmers [C. G. Khoury, S. J.Norton, T. Vo-Dinh, “Plasmonics of 3-D Nanoshell Dimers Using MultipoleExpansion and Finite Element Method ACS Nano, 3, 2776-2788 (2009)], andmulti-layer metallic nanoshells [S. J. Norton, T. Vo-Dinh, “Plasmonicsenhancement of a luminescent or Raman-active layer in a multilayeredmetallic nanoshell”, Applied Optics, 48, 5040-5049 (2009)]. The entirecontents of each of the above noted references in this paragraph areincorporated herein by reference. In various embodiments of theinvention, multi-layer metallic nanoshells discussed in this applicationhave the potential capability to enhance electromagnetically twospectral regions. Accordingly, the metallic structures of the inventioncan be used in the upconverting mode to enhance both the excitation atwavelength λ₁ and the emission at wavelength λ₂ This feature also can beused in the down converting to enhance primarily the emission atwavelength λ₂ and potentially the excitation at wavelength λ₁.

Such metallic structures in various embodiments of the invention includeconducting materials made for example of metals, or doped glasses ordoped semiconductors. These conducting materials can be in the form ofpure or nearly pure elemental metals, alloys of such elemental metals,or layers of the conducting materials regardless of the constituency.The conducting materials can (as noted above) include non-metallicmaterials as minor components which do not at the levels ofincorporation make the composite material insulating.

Similarly, in various embodiments of the invention, the up or downconverting materials can include at least one of a dielectric, a glass,or a semiconductor. The up or down converting materials can include analloy of two or more dielectric materials, an alloy of two or moreglasses, or an alloy of two or more semiconductors.

Accordingly, FIG. 10A represents embodiments of the invention where thedielectric core is supplemented with a shell. The shell can include ametal layer of a prescribed thickness. The metal layer can includematerials such as nickel, gold, iron, silver, palladium, platinum andcopper and combinations thereof. The shell functions as a plasmonicshell where surface plasmons can form in the metal between thedielectric core and the outer environment acting as an exteriordielectric. The shell (as shown) may not be a complete shell. Partialmetallic shells or metallic shells of varying thicknesses are alsoacceptable in the invention.

FIG. 10B shows yet other embodiments of upconversion structures thathave a dielectric layer between the metal and the UC materials.

FIG. 10C shows still further embodiments of plasmonics-activenanostructures having upconverting (UC) materials that can be designed:(a) a metal nanoparticle, (b) an UC nanoparticle core covered with metalnanocap, (c) a spherical metal nanoshell covering an UC spheroid core,(d) an oblate metal nanoshell covering UC spheroid core, (e) a metalnanoparticle core covered with UC nanoshell, (f) a metal nanoshell withprotective coating layer, (g) multi layer metal nanoshells covering anUC spheroid core, (h) multi-nanoparticle structures, (i) a metalnanocube and nanotriangle/nanoprism, and (j) a metal cylinder.

FIG. 10D shows yet other embodiments of plasmonics-active nanostructureshaving upconverting materials with linked photo-active (PA) moleculesthat can be designed. For example, for the case of psoralen (as the PAmolecule), the length of the linker between the PA molecule and the UCmaterial or the metal surface is tailored such that it is sufficientlylong to allow the PA molecules to be active (attach to DNA) and shortenough to allow efficient excitation of light from the UC to efficientlyexcite the PA molecules. FIG. 10D shows (a) PA molecules bound to an UCnanoparticle, (b) an UC material-containing a nanoparticle covered withmetal nanoparticles, (c) a metal nanoparticle covered with UC materialnanocap, (D) an UC material-containing nanoparticle covered with metalnanocap, (e) a metal nanoparticle covered with an UC material nanoshell,(f) an UC material-containing nanoparticle covered with metal nanoshell,(g) an UC material-containing nanoparticle covered with metal nanoshellwith protective coating layer.

IR frequencies have significant penetration into the human body andpermit the primary excitation λ₁ to penetrate subcutaneously into thebody tissue. Upon their penetration into the body tissue, the dielectriccore of the invention interacts with the incident radiation λ₁ togenerate the secondary light λ₂ as described above. Therefore,permitting the generation in situ to the body of a wavelength λ₂ whichmay be in the UV or visible range is appropriate for activations ofpsoralen or other types of drugs known to be activated by a UV orvisible light source.

Since the dielectric cores of this invention have the ability to beselectively stimulated by discrete wavelengths of and produce discreteemission wavelengths at λ₂, the medial applications can be manipulatedso that a number of dual purpose diagnostic/treatment tools can beproduced.

For example, in one embodiment of the invention, a material such as theabove-described co-doped yttrium oxide is introduced into the body.Yttrium oxide as a host is known to be a down converter from X-rayradiation. In this particular example, X-ray incident radiation on theyttrium oxide will produce UV light which would in turn be used toactivate drugs such as psoralen for the treatment of cancer. Meanwhile,the co-doped yttrium oxide as a upconverter could be used where the NIRexcitation could produce an emission at a wavelength that was differentthan that produced from the X-ray down conversion radiation. In thismanner, the progression of the yttrium oxide (with drug attached as therecipient 4) into a target organ to be treated could be monitored usingthe NIR light as the excitation source and collecting the visible lightin some type of CCD camera. Once the yttrium oxide particles wereabsorbed into the respective tumor cells for treatment, at that point intime, X-ray radiation could be initiated and thereby activating thepsoralen tagged yttrium oxide and providing an effective means fortreating the tumor cell.

FIG. 23 is a depiction of down conversion and up conversion emissionfrom a NaYbF₃; Tm nanoparticle. The up conversion lines were excited at980 nm. The down conversion lines were excited with 320 kV x-rays. FIG.24 is a micrograph of a representative 35 nm PEI Coated YbF₃; Tm (2%)particle.

Alternatively, in another dual purpose diagnostic/treatment example, onecan choose a system where the NIR wavelength is specifically tuned fordiagnostics as explained above while excitation with a separatewavelength of NIR can be used to produce UV light (through anotherupconversion channel) that would itself activate a recipient molecule(e.g. psoralen for cancer treatment) without the necessity of X-ray anddown conversion activation. This feature then permits one to use a drugwhich either would be acceptable for deep body penetration through X-rayradiation or would be acceptable for more shallow body penetrationthrough NIR radiation to treat cancer cells that were located indifferent parts of the body relative to the surface of the body.Moreover, fiber optics could be used to direct the NIR light (through asurgical incision for example) directly to a target. By locallyactivating the psoralen and by the known autovaccine effect, thisinitially local NIR activated treatment may be effective at treatingcancer outside the NIR irradiated area.

Examples of such dual use drugs which all exhibit NIR activation andupconversion for the purpose of imaging and/or to excite psoralen wouldinclude the dual dopants of yttrium oxide, the dual dopants of neodymiumoxide, triply doped ytterbium thulium neodymium oxides, the dual dopantsof sodium yttrium fluoride, and the dual dopants of lanthanum fluoride.For example, by providing a ytterbium-thulium doped yttrium oxidecontaining 95% verses 5% dopant concentration with another lanthanide,one will produce diagnostic/treatment functions through pure NIRexcitation, having the drug treatment excitable at 980 nanometers versesthe diagnostic imaging process excitable at 808 nanometers withdifferent emissions coming from each excitation process.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Gold Nanoshell Preparations with Dielectric Cores:

The present invention can utilize a wide variety of synthesizedmetallic-coated core-shell nanoparticles prepared from a number of wetchemical procedures. The techniques described below are provided for thepurposes of illustration and not for the purpose of limiting theinvention to these particular techniques. In the present invention, goldnanoshells can be prepared using the method or similar methods describedin Hirsch L R, Stafford R J, Bankson J A, Sershen S R, Rivera B, Price RE, Hazle J D, Halas N J, West J L (2003) Nanoshell-mediated nearinfrared thermal therapy of tumors under MR Guidance. Proc Natl Acad Sci100:13549-13554, the entire contents of which are incorporated herein byreference. This method uses a mechanism involving nucleation and thensuccessive growth of gold nanoparticles around a dielectric core.Dielectric nanoparticles of sizes less than for example 100, 200, or 300nm, as well as larger sizes, used for the core of the nanoshells, canthen be monodispersed in a solution of 1% APTES in EtOH. The gold “seed”colloid can then be synthesized using the Frens method (see detailsbelow) and deposited onto the surface of the dielectric nanoparticlesvia molecular linkage of silyl terminated amine groups. The gold “seed”covers the aminated nanoparticle surface, first as a discontinuous goldmetal layer and gradually growing forming a continuous gold shell.

Additionally, various photochemical methods have been reported for thefabrication of gold nanoparticles and gold films [Refs: A. Pal, T. Pal,D. L. Stokes, and T. Vo-Dinh, “Photochemically prepared goldnanoparticles: A substrate for surface-enhanced Raman scattering”,Current Science, 84, 1342-1346 (2003; A. Pal, D. L. Stokes and T.Vo-Dinh, “Photochemically Prepared Gold Metal film in aCarbohydrate-based Polymer: a Practical Solid substrate forSurface-enhanced Raman Scattering, Current Science, 87, 486-491 (2004)].These articles in their entirety are incorporated herein by reference.The present invention in various embodiments utilizes a class ofcore-shell nanoparticles based on rare earth oxide (REO) cores havingnoble metal shells. A number of nanoparticle/metal shell systems can befabricated using the photochemical procedures described below or othersuitably modified procedures.

The REO core material is a well-suited core material for the presentinvention due to doping for either upconversion- or downconversion-basedfluorescence, and due to the fact that the plasmonically-active metalshells can be easily functionalized with targeting peptides,fluorophores, or SERS-active molecules using well-establishedtechniques. For the purpose of illustration, the design and fabricationof one such hybrid nanoparticle system is described below where thenanoparticle system includes an yttrium oxide (Y₂O₃) core, a gold (Au)shell, and a short arginine and lysine-rich peptide, e.g.,transactivator of transcription (TAT) residues 49-57, functionalizedwith various fluorescent dyes using N-hydroxysuccinimide (NHS) couplingchemistry. This peptide and similar molecules can show greatly enhancedcellular uptake and nuclear localization of DNA, nanoparticles,liposomes, peptides and proteins. Further, this particular portion ofthe TAT sequence has been shown to be non-toxic, making the resultingfluorescently-labeled nanoparticles potentially suitable for in vivoimaging applications.

Materials:

Yttrium oxide nanoparticles (e.g., 99.9% purity, 32-36 nm averagediameter, cubic crystal structure) were obtained from Nanostructured andAmorphous Materials, Inc. (Houston, Tex.). Tri-arginine(H-Arg-Arg-Arg-OH) acetate was obtained from Bachem (Torrance, Calif.),and gold tribromide (AuBr₃) was obtained from Alfa Aesar (Ward Hill,Mass.). Dimethyl sulfoxide (DMSO) was purchased from CalBioChem (LaJolla, Calif.) and was used as received. A cysteine-modified version ofthe TAT peptide (residues 49-57, sequenceArg-Lys-Lys-Arg-Arg-Arg-Gln-Arg-Arg-Cys-CONH₂ (SEQ ID NO: 1), molecularweight 1442 g/mol, hereafter referred to as “TAT”) was obtained fromSynBioSci (Livermore, Calif.).Succinimidyl-[4-(psoralen-8-yloxy)]butyrate (SPB) was obtained fromPierce (Rockford, Ill.), and Marina Blue, Alexa 350 and Alexa 546 NHSesters were obtained from Invitrogen (Carlsbad, Calif.). Ultrapure 18.2Mf deionized (DI) water purified with a Millipore Synergy filtrationsystem (Millipore, Billerica, Mass.) was used to make all solutions.

Yttrium Oxide Dispersion:

Tip sonication was used to disperse autoclaved Y₂O₃ nanoparticles at 10mg/mL in 10 mM tri-arginine solution which had been pre-filtered at 0.22microns. Following moderate mixing in a sealed, sterile container on astir plate for 24 hours to allow tri-arginine attachment and improvedY₂O₃ dispersion, the solution was centrifuged at 8200 relativecentrifugal force (RCF) to remove fused particles and large aggregates.

Gold Shell Formation:

Supernatant from the initial Y₂O₃ dispersion was diluted 1:1 (v/v) with5.7 mM AuBr₃ dissolved in sterile DI water and pre-filtered at 0.22microns, then exposed to high-intensity fluorescent light (CommercialElectric, Model 926) for 16 hours in a sealed, sterile glass containerwith moderate mixing. During the time course of this photochemicalprocess, the reddish-brown AuBr₃ solution turned yellow immediatelyafter addition of the Y₂O₃ in tri-arginine; became clear and visuallycolorless; then developed an intense purple color as Au shells formed onthe Y₂O₃ cores. In the absence of the Y₂O₃ cores, neither the intensepurple color associated with plasmonic absorption by gold nanoshells northe deep red color associated with solid gold nanoparticles appears. Useof heat rather than light in the presence of Y₂O₃ particles tends toproduce a large number of solid gold nanoparticles rather than or inaddition to core-shell structures, as evidenced by strong absorption at˜530 nm.

Particle Functionalization with TAT:

Gold-coated Y₂O₃ nanoparticles were centrifuged at 16k RCF for 15minutes, and the pellet was re-dispersed in a 50% volume of sterile DIwater by a short tip sonication. The particles were further purified bytwo additional centrifugations at 16k RCF for 15 minutes each, withredispersion in a 100% volume of sterile DI water following the secondcentrifugation and final redispersion in a 100% volume of 1 mg/mL (0.7mM) TAT peptide dissolved in sterile DI water and pre-filtered at 0.22microns.

This solution was vigorously mixed at room temperature for one hour toallow thiol anchoring to the gold shell via the c-terminal cysteineresidue. Variations in the TAT concentration, temperature and reactiontime can all change the extent of surface coverage and the potential forfurther functionalization.

Peptide Functionalization with Dye Molecules:

The TAT-functionalized, gold-coated Y₂O₃ particles were purified bytriplicate centrifugation at 16k RCF, with the first two re-dispersionsin sterile DI water and the final re-dispersion in sterile 100 mMbicarbonate buffer at pH 9.0. Each NHS ester (SPB, Alexa 350, MarinaBlue and Alexa 546) was dissolved at 10 mg/mL in dimethyl sulfoxide(DMSO), and 100 microliters of a given NHS-functionalized dye were addedto a 1 mL aliquot of TAT-functionalized, gold-coated Y₂O₃. The solutionswere reacted for one hour at room temperature in the dark with vigorousmixing to allow attachment of dye molecules to primary amines along theTAT peptide (such as the attachment of N terminus and the lysine sidechains).

The psoralen-functionalized nanoparticles were centrifugally cleanedusing a 1:1 volume of DMSO in water to remove any residual SPB crystals,then all dye-functionalized core-shell nanoparticles were purified bytriplicate centrifugation at 16k RCF for 15 minutes. Each centrifugationstep was followed by re-dispersion in a 100% volume of sterile DI water.Presuming removal of 95+% of non-attached dye molecules during eachcentrifugation step, no more than 0.01% of the unbound dye is estimatedto remain in the final solution.

Nanoparticle Characterization:

Transmission electron microscopy (TEM) provides additional evidence forthe presence of gold-coated Y₂O₃ particles. FIG. 10E, for example, showsa representative TEM image of as purchased Y₂O₃ nanoparticles. Theparticles are quite polydisperse, but exhibit an average diameter ofapproximately 35 nm. FIG. 10F shows similar images for Y₂O₃ particlescoated with a gold shell using the synthetic procedure described above.Like the underlying Y₂O₃ cores, the gold-coated yttrium oxide particlesare somewhat polydisperse with an average diameter of approximately 50nm.

Perhaps the most conclusive demonstration that these nanoparticles arein fact gold-coated Y₂O₃ comes from comparison of X-ray diffraction data(XRD). FIG. 10G shows diffractograms for both the initial cubic Y₂O₃nanoparticles (lower trace) and the final gold-coated core-shellparticles (upper trace). Strong peaks at 2θ=29, 33.7, 48.5 and 57.5degrees in the lower trace are indicative of cubic Y₂O₃. The mostpronounced features in the upper trace are two gold-associated peaks at2θ=38.2 and 44.4 degrees. In addition, the four strongest cubic Y₂O₃peaks at 2 0=29, 33.7, 48.5 and 57.5 degrees are also visiblysuperimposed on the baseline diffractogram from the gold nanoshells. Thereason for the broadening of the Y₂O₃ peak at 2θ=29 degrees is notdefinite, but may be a result of gold-Y₂O₃ interactions or,alternatively, the preferential size-selection of small Y₂O₃ particlesduring the 8200 RCF centrifugation used to remove large Y₂O₃ particlesand aggregates.

Gold Colloidal Nanoparticles:

In various embodiments of the present invention, gold nanoparticleswithout a dielectric core are used in the medium being irradiated toenhance either the intensity of the initiation energy (i.e., the primarysource: for example an IR laser for upconversion or an xray beam fordown conversion) or to enhance the light generated from the upconvertingor down converting nanoparticles). The techniques described below forthe fabrication of metal nanoparticles with and without cores and withand without additional layers and linkageas are provided for thepurposes of illustration and not for the purpose of limiting theinvention to these particular techniques. Indeed, the present inventioncan utilize a wide variety of synthesized metallic, multi-layercore-shell nanoparticles prepared from a number of wet chemicalprocedures. Exemplary parameters and procedures for producing thesenanoparticles systems are described below. Starting materials includedultrapure water (deionized), HAuCl₄*3H₂O, AgNO₃, Y₂O₃, NaOH, NH₄OH,sodium citrate, hydroxylamine hydrochloride, hydrazine monohydrate,sodium borohydride, aminopropyltrimethoxy silane (APTMS), sodiumsilicate, tetraethyl orthosilicate (TEOS), methanol, ethanol,isopropanol, oleic acid, and oleylamine.

a. Synthesis of Gold Nanoparticles

The Frens method (see G. Frens, Nat. Phys. Sci. 241 (1973) 20, theentire contents of which are incorporated herein by reference) can beused to synthesize gold nanoparticles. In this process, 5.0×10⁻⁶ mol ofHAuCl₄ was dissolved in 19 mL of deionized water. The resulting solutionwas faintly yellow. The solution was heated and vigorously stirred in arotary evaporator for 45 minutes. One mL of 0.5% sodium citrate wasadded, and the solution was stirred for an additional 30 minutes.Addition of sodium citrate has multiple purposes. First, citrate acts asa reducing agent. Second, citrate ions that adsorb onto the goldnanoparticles introduce surface charge that stabilizes the particlesthrough charge repulsion, thus preventing nanocluster formation.

b. Synthesis of Gold Nanoparticles having 15-nm Diameter

Two mL of 1% gold chloride in 90 mL DI water was heated to 80° C. for 15minutes, then 80 mg sodium citrate in 10 ml DI water was added. Thesolution was boiled and vigorously stirred for 30 minutes. FIG. 10Hshows pictures of ˜15-nm gold nanoparticles prepared using citratereduction.

c. Synthesis of 30-nm Gold Nanoparticles

Two mL of 1% HAuCl₄ solution in a 100-mL round-bottom flask were mixedwith 20 mg of sodium citrate, then boiled and vigorously stirred for 30minutes. FIG. 10I shows TEM images of 30-nm gold nanoparticles preparedusing the citrate reduction technique.

d. Synthesis of 60-nm Gold Nanoparticles

Two mL of 1% HAuCl₄ in 100 mL of water were mixed with 10 mg of sodiumcitrate. The solution was boiled and vigorously stirred for 30 minutes.FIG. 10J shows TEM pictures of 60-nm gold nanoparticles prepared usingthe citrate reduction technique.

e. Use of Hydrazine Monohydrate as a Reducing Anent:

100 microliters (0.1 mL) of 12 millimolar gold chloride solution wasdiluted with 80 ml H₂O in a beaker. The initial pH of the gold solutionwas 3.67. The temperature of the solution was increased to 80° C. for 30minutes, at which point 0.3 mL hydrazine monohydrate was added to thegold solution. The solution pH shifted to 7.64. Over time, gold solutionchanged to a very light pink color. FIG. 10K shows TEM pictures of˜30-nm gold nanoparticles prepared using the hydrazine monohydratereduction technique.

Colloidal Silver Nanoparticles:

Silver nanoparticles, like the gold nanoparticles described above, canbe used in the present invention to enhance either the intensity of theinitiation energy (i.e., the primary source: for example an IR laser forupconversion or an X-ray beam for down conversion) or to enhance thelight generated from the upconverting or down-converting nanoparticles).Silver nanoparticles have been prepared from AgNO₃ using a variety ofreducing agents. FIG. 10L shows a TEM image of silver nanoparticlesprepared using the procedures described below.

Use of Sodium Citrate as a Reducing Agent:

In this method, 50 mL of a 10⁻³ M AgNO₃ aqueous solution was heated toboiling. Then, 1 mL of a 1% trisodium citrate (C₆H₅O₇Na₃) was added tothe solution, and the solution was maintained at boiling for 1 hourbefore being allowed to cool. The resultant colloidal mixture exhibiteda dark grey color.

Use of Hydroxylamine Hydrochloride as a Reducing Agent:

A colloidal solution was formed by dissolving 0.017 g of silver nitrate(AgNO₃) in 90 mL water. 21 mg of hydroxylamine hydrochloride (NH₃OH.HCl)was dissolved in 5 mL water and 4.5 ml of 0.1 M sodium hydroxide wasadded. This mixture was added to the AgNO₃ solution. In just in a fewseconds, a grey-brown solution appeared.

Use of Sodium Borohydride as a Reducing Agent:

Aqueous solutions containing 10 mL 10⁻³ M AgNO₃ and 30 mL 10⁻³ M NaBH₄were mixed under ice-cooled conditions. The AgNO₃ solution was addeddropwise to the NaBH₄ solution with vigorous stirring. The resultantmixture was allowed to age 1 hour before stirring the resultant mixtureagain for 10 minutes.

Metallic/Dielectric, Multi-Layer, Core-Shell Nanoparticles:

As seen in FIGS. 10A-10D, the present invention in various embodimentsutilizes multilayer dielectric/metal structures.

Au Nanoparticles Coated with Ag or Ag Nanoparticles Coated with Au:

Core-shell nanoparticles such as gold-coated silver nanoparticles andsilver-coated gold nanoparticles have been synthesized in an aqueousmedium using CTAB as a surfactant and ascorbic acid as a reducing agentCore nanoparticles (i.e. Au or Ag) were prepared using the aboveprocedures, then coated with secondary, tertiary, etc. shells.

For example, spherical gold nanoparticles (˜15 nm) were prepared byboiling HAuCl₄ in the presence of sodium citrate. For coating gold witha layer of silver, 1 mL of 0.1 M ascorbic acid solution, 0.5 mL of 10 mMAgNO₃ solution, and 0.5 mL of the previously formed Au colloid weresequentially added to 20 mL of a 50 mM CTAB solution. Subsequently, 0.1mL of 1.0 M NaOH was added dropwise, which led to a fast color change(from red to yellow). FIG. 10M shows TEM images of Au nanoparticlescoated with Ag.

A similar procedure was used to prepare Ag nanoparticles coated with Au.The use of solutions of a mixture of AgNO₃ and HAuCl₄ would yield analloy of Ag and Au.

Au@Ag@Au@Ag Multi Shell Nanoparticles:

Multishell nanoparticles such as Au@Ag@Au@Ag were prepared using CTAB asa surfactant, and ascorbic acid and NaOH as reducing agents. Sphericalgold nanoparticles (˜15 nm) were prepared by boiling HAuCl₄ in thepresence of sodium citrate. To coat gold cores with a layer of silver,20 mL of a 50 mM CTAB, 1 mL of 0.1 M ascorbic acid, 0.5 mL of 10 mMAgNO₃, and 0.5 mL of the Au colloid were sequentially mixed.Subsequently, 0.1 mL of 1.0 M NaOH was added in a dropwise manner, whichled to a fast color change from red to yellow.

Then, another gold layer was coated by mixing 20 mL of the Ag-coated Aucolloid in water with 1 mL of the ascorbic acid solution. The resultingmixture was then added to 0.05 mL of 0.10 M HAuCl₄ in a dropwise manner.The solution color changed to deep blue at this stage. Subsequently, anouter silver shell was formed on the previously formed Au@Ag@Aunanoparticles by mixing 20 mL of colloid with 0.5 mL 10 mM AgNO₃followed by drop wise addition of 0.2 mL of 1.0 M NaOH. The solutionthen showed a color change to orange. FIG. 10N shows TEM images ofAu@Ag@Au@Ag multi-shell nanoparticles.

All of the above core-shell nanoparticle solutions were stable insolution.

Y₂O₃ Coated with SiO₂, Y₂O₃ Coated with Au, Y₂O₃ Coated with Ag or AuCoated SiO₂ Coreshell Nanoparticles:

Procedures similar to those used in the preparation of core-shell goldor silver nanoparticles can be employed to synthesize Y₂O₃ coated withAu or Y₂O₃ coated with Ag.

Metal (Au Coated with SiO₂) or REO Nanoparticles Coated with SiO₂:

SiO₂ can be coated on gold, silver and REO nanoparticles. There arevarious procedures available in the literature. See for example W.Stöber, A. Fink, E. Bohn, J. Colloid Interface Sci. 26 (1962) 62-69; Y.Kobayashi, H. Katakami, E Mine, D. Nagao, M Konno, L. M Liz-Marzdn,Journal of Colloid and Interface Science 283 (2005) 392-396; L. MLiz-Marzán, M Giersig and P. Mulvaney, Langmuir 1996, 12, 4329-4335; S.P. Mulaney, M. D. Musick C. D. Kearting, M. J. Natan, Langmuir 2003, 19,4784-4790; Q. Lu, A. Li, F. YunGuo, L. Sun and L. C. Zhao,Nanotechnology 19 (2008) 205704; Jana, et. al., Chem. Mater., Vol. 19,p. 5074-5082 (2007), the entire contents of each of these references areincorporated herein by reference. In this silica-coating method, whichinvolves condensation of alkoxysilanes on the nanoparticle surface,various types of functional silanes which have alcoxysilyl groups (e.g.,methoxysilyl, ethoxysilyl, isopropoxysilyl, etc.) at one end and anamino or thiol group at the other end are typically used. It has beenshown that alcoxysilyl groups undergo hydrolysis in a basic or acidicmedium to form a silica shell.

The present invention employs two different strategies to induce silicapolymerization on the nanoparticle surface. In the case of REOnanoparticles, the silanization process involves condensation of silaneswith the hydroxyl groups on the REO particle surface. For Au and Ag,mercapto or amino silane can be used as a linker. In this case, thethiol group of this linker silane chemisorbs onto the metal nanoparticlesurface, and the alcoxysilane groups initiate silica shell formation onthe nanoparticle surface.

Optimization of the silanization conditions has been performed in orderto fabricate water-soluble nanoparticles. There are in general twoprimary steps in the silane conjugation scheme. First, it is importantthat excess ligands be removed from the starting nanoparticles. Second,temperature, heating time, and pH all play critical roles in the rate ofsilane hydrolysis. Both alkyl amines and aminosilane, for example, canserve as a base for the catalytic hydrolysis of alkoxysilane at 65-70°C. In some procedures, nanoparticle-silane conjugates begin toprecipitate within 3-5 min of reaction, and finish within 15-30 min. Ifa specific shell thickness is desired, the hydrolysis can be stopped atany time by quenching the reaction to room temperature or by separatingthe precipitate from the solution. This is useful because furtherheating of the precipitated nanoparticle-silane conjugates withoutseparating them from free silanes can produce interparticlecross-linking via hydrolysis. If excess precursor is removed,intra-particle crosslinking can proceed without the potential ofinterparticle cross-linking.

Chemical Synthesis of Multi-Layer Core-Shell Structures Using Y₂O₃

To deposit multiple shells on Y₂O₃ nanoparticles, Y₂O₃ nanoparticleswere initially coated with Ag via UV photoreduction in a proceduresimilar to that discussed above for gold shells. In the presentinvention, a number of approaches can be utilized for the addition of agold shell. These include 1) a sodium citrate process, 2) a sodiumborohydride reduction, 3) a hydrazine monohydrate reduction, 4) asolution containing hydroxyl amine and NaOH, and 5) a mixture of CTAB,ascorbic acid, and NaOH.

Use of Sodium Citrate as a Reducing Agent:

A typical experiment used 0.1 to 1 mL of Y₂O₃ coated with Ag (˜50 nm), 1to 3 mL of 2.5 H10⁻³ M HAuCl₄, and 50 mL distilled water in a 100 mlround bottom flask. This solution was boiled with constant stirring, and3 mL of 1 wt % sodium citrate was added. The resultant colloidalsolution color became black with a pH of approximately pH 6.5. Thesolution was stirred for another 15 min and then allowed to stand.

Use of Sodium Borohydride as Reducing Agent:

A typical experiment used 0.1 to 1 mL of Y₂O₃ coated with Ag (˜50 nm), 1to 3 mL of 2.5 H10⁻³ M HAuCl₄, and 50 mL distilled water in a 100 mLround bottom flask. Under constant stirring this solution was boiledprior to addition of 0.1 to 1 mL of 0.1 M NaBH₄ solution. The resultantcolloidal solution became black and aggregated within a few minutes.

These fabrication procedures provide the present invention with a numberof nanoparticle systems for application to a variety of media ormaterials where the nanoparticles can directly or indirectly generatelight from an initiation energy or enhance the generated light or theradiation initiation energy.

In a further embodiment of the invention, the upconverter structures ofthe invention can be incorporated into a material (e.g., biocompatiblepolymer) that can form a nanocap onto the metal (gold) nanoparticles.The material can be a gel or biocompatible polymer that can havelong-term continuous release properties.

Suitable gel or biocompatible polymers include, but are not limited topoly(esters) based on polylactide (PLA), polyglycolide (PGA),polycarpolactone (PCL), and their copolymers, as well aspoly(hydroxyalkanoate)s of the PHB-PHV class, additional poly(ester)s,natural polymers, particularly, modified poly(saccharide)s, e.g.,starch, cellulose, and chitosan, polyethylene oxides, poly(ether)(ester)block copolymers, and ethylene vinyl acetate copolymers.

In a further embodiment, the metallic nanoparticles without a dielectriccore can be provided in the medium along with the upconvertingmetal-covered dielectric core nanoparticles so that the “pure” metallicnanoparticles can enhance interaction of the upconverted light withanother agent or recipient in the medium (such as for example aphotosensitizer, a photoactivatable drug, or a photoinitiator).

FIG. 11 shows other possible embodiments where a recipient molecule isbound to the metal nanoparticles via a linker that can be cut by photonradiation. Such a linker includes, but is not limited to, a biochemicalbond, a DNA bond, an antibody-antigen bond, or other bond which, whenexcited by light, reorganizes its bonding electrons to non- oranti-bonding state. In another embodiment, the linker is a chemicallylabile bond that will be broken by the chemical environment inside thecell. In various embodiments, it may be more difficult for metalnanoparticles to enter targeted sites in the medium than for smallermolecules.

Aggregation of metal (such as silver or gold) nanoparticles(nanospheres, nanorods, etc) is often a problem, especially withcitrate-capped gold nanospheres, cetyl trimethylammonium bromide(CTAB)-capped gold nanospheres, nanorods, and nanoshells because theyhave poor stability when they are dispersed in buffer solution due tothe aggregating effect of salt ions. The biocompatibility can beimproved and nanoparticle aggregation prevented by capping thenanoparticles with polyethylene glycol (PEG) (by conjugation ofthiol-functionalized PEG with metal nanoparticles).

The majority of immobilization schemes involving metal surfaces, such asgold or silver, utilize a prior derivatization of the surface withalkylthiols, forming stable linkages. Alkylthiols readily formself-assembled monolayers (SAM) onto silver surfaces in micromolarconcentrations. The terminus of the alkylthiol chain can be used to bindbiomolecules, or can be easily modified to do so. The length of thealkylthiol chain has been found to be an important parameter, keepingthe biomolecules away from the surface, with lengths of the alkyl groupfrom 4 to 20 carbons being preferred.

There are many methods related to the preparation of stableoligonucleotide conjugates with gold particles by usingthiol-functionalized biomolecules that have previously been shown toform strong gold-thiol bonds. These methods described below can be usedin various embodiments of the invention. Oligonucleotides with5′-terminal alkanethiol functional groups as anchors can be bound to thesurface of gold nanoparticles, and the resulting labels were robust andstable to both high and low temperature conditions [R Elghanian, J. J.Storhoff R. C. Mucic, R. L. Letsinger and C. A. Mirkin, Selectivecolorimetric detection of polynucleotide based on the distance-dependentoptical properties of gold nanoparticles. Science 277 (1997), pp.1078-1081], the entire contents of which are incorporated herein byreference. A cyclic dithiane-epiandrosterone disulfide linker has beendeveloped for binding oligonucleotides to gold surfaces. Id Li et al.have reported a trithiol-capped oligonucleotide that can stabilize goldmetal nanoparticles having diameters=100 nm, while retaininghybridization properties that are comparable to acyclic ordithiol-oligonucleotide modified particles [Z. Li, R. C. Jin, C. A.Mirkin and R. L. Letsinger, Multiple thiol-anchor capped DNA-goldnanoparticle conjugates. Nucleic Acids Res. 30 (2002), pp. 1558-1562],the entire contents of which are incorporated herein by reference.

In general, silver nanoparticles can not be effectively passivated(i.e., made less reactive) by alkylthiol-modified oligonucleotides usingthe established experimental protocols that were developed for goldparticles. One method of generating core-shell particles having a coreof silver and a thin shell of gold has allowed silver nanoparticles tobe readily functionalized with alkylthiol-oligonucleotides to preparepure gold particle-oligonucleotide conjugates, suitable in variousembodiments of the invention. [Y. W. Cao, R Jin and C. A. Mirkin,DNA-modified core-shell Ag/Au nanoparticles. J. Am. Chem. Soc. 123(2001), pp. 7961-7962], the entire contents of which are incorporatedherein by reference.

Silver surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 150. There is also a larger thiol packing density on silver,when compared to gold. See Burges, J. D.; Hawkridge, F. M. in Langmuir1997, 13, 3781-6, the entire contents of which are incorporated hereinby reference. After self-assembled monolayer (SAM) formation ongold/silver nanoparticles, alkylthiols can be covalently coupled tobiomolecules. The majority of synthetic techniques for the covalentimmobilization of biomolecules utilize free amine groups of apolypeptide (enzymes, antibodies, antigens, etc) or of amino-labeled DNAstrands, to react with a carboxylic acid moiety forming amide bonds.

Such bonding schemes have applications not only by providing a mechanismby which the nanoparticles can be controllably dispersed and deliveredwithin a medium, but may also play a role in the formation ofencapsulated upconverter structures of the invention.

With the upconverter structures of the invention, a plasmonics effect isadvantageous. A plasmonics effect can occur throughout theelectromagnetic region provided the suitable nanostructures, nanoscaledimensions, metal types are used. Plasmonic effects are possible over awide range of the electromagnetic spectrum, ranging from gamma rays andX rays throughout ultraviolet, visible, infrared, microwave and radiofrequency energy. However, for practical reasons, visible and NIR lightare used for metal structures such as for example silver and goldnanoparticles, since the plasmon resonances for silver and gold occur inthe visible and NIR region, respectively. Especially for goldnanoparticles, the NIR region is very appropriate for the delivery ofenergy into a medium where otherwise optical scatter at shorterwavelengths would present a problem, such as for example in thetreatment of waste water or the sterilization of food products havinghigh concentrations of suspended solids or the delivery ofphotoactivatible drugs into a living cell.

The invention includes several methods for using light to excitephotoactivate or photostimulate compounds in the medium. Light havingwavelengths within the so-called “window” (designed to penetrate anycontainer holding the medium to be processed and/or to transmit throughthe medium) can be used. Moreover, while certain aspects of theinvention prefer that the excitation light be nominally non-absorbing(or nearly transparent) in the medium, due to the plasmonic advantages,the invention is still useful in mediums even when there is considerablescatter and absorption.

The ability of light to penetrate the medium depends on absorption andscatter. Within the hydrous medium, a window extends from 600 to 1300nm, from the orange/red region of the visible spectrum into the NIR. SeeT. Vo-Dinh, Biomedical Photonics Handbook, CRC, 2003. At theshort-wavelength end, absorbing biomolecules become important, includingDNA and the amino acids tryptophan and tyrosine. At the infrared (IR)end of the window, penetration is limited by the absorption propertiesof water (water vibrational overtone absorptions start to becomeimportant at 950 nm). Within the window, scattering is dominant overabsorption, and so the propagating light becomes diffuse, although notnecessarily entering into the diffusion limit.

In various embodiments of the invention, the upconverter structures arecovered with a layer (1-30 nm) of dielectric material (e.g. silica orpolymer). The dielectric layer (or nanoshell) is designed to preventquenching of the luminescence light emitted from a dielectric core(e.g., a La doped-dielectric core). Quenching can sometimes occur due todirect contact of a metal to the receptor or media. To address thisissue, recipient molecules are bound to (or in proximity of) the metalshell via a spacer (linker). The spacer is designed to prevent quenchingof the luminescence light emitted by the dielectric core.

FIG. 12A shows an embodiment of the present invention where thedielectric core has appended thereon or attached by linkages a recipientmolecule such as a photo-active molecule. An appended molecule is onethat is typically directly bonded either by a covalent bond or a dativeassociation. Linkers are typically added to covalently tether themolecule to the nanocrystal. In various embodiments of the presentinvention, either mechanism can be used to secure the recipientmolecule. The photo-active molecule 6 is receptive to interaction withthe generated light A such that upon interaction with the light λ₂chemical reactions or pharmaceutical reactions are induced therein orthere from. For example, UV light generated from the upconverterstructures can either change the state of the photo-active molecule to areactive state, or can sever the linkages releasing the recipientmolecule 6 into the medium. As shown in FIG. 12A, in one embodiment ofthe invention, the upconverter material is itself separated from a metalcomponent. The exact distances between the recipient molecule and thedielectric core can be varied by using certain chemical linkingcompounds and as explained below that may also provide certain steric orsynergistic effects.

As shown in FIG. 12A, in one embodiment of the invention, the recipientmolecule can be a bioreceptor. Bioreceptors are the key to specificityfor targeting disease cells or mutate genes or specific biomarkers. Theyare responsible for binding the biotarget of interest to the drug systemfor therapy. Bioreceptors can take many forms and the differentbioreceptors that have been used are as numerous as the differentanalytes that have been monitored using biosensors. However,bioreceptors can generally be classified into different majorcategories. These categories include: 1) antibody/antigen, 2) enzymes,3) nucleic acids/DNA, 4) cellular structures/cells, 5) peptides, 6)saccharides, and 5) biomimetic. FIG. 8A illustrates various upconversionstructures with bioreceptors that can be designed. The probes aresimilar to those previously described but also have a bioreceptor fortumor targeting. Accordingly, in one embodiment of the presentinvention, the upconversion structures include (a) photoactive (PA)molecules bound to a metal nanoparticle having a bioreceptor, (b)PA-linked UC material nanoparticle covered with metal nanoparticles,having a bioreceptor, (c) a metal nanoparticle covered with an UCmaterial nanocap with linked PA molecule, having a bioreceptor, (d) anUC material nanoparticle covered with metal nanocap and linked PA,having a bioreceptor, (e) a metal nanoparticle covered with an UCmaterial nanoshell with PA, having a bioreceptor, (f) an UC materialnanoparticle covered with metal nanoshells, having a bioreceptor, (g) anUC material nanoparticle covered with a metal nanoshell with aprotective coating layer, having bioreceptor.

FIG. 12B shows yet other embodiments of plasmonics-active nanostructureshaving upconverting material (UC) with linked photo-active (PA)molecules and also having a bioreceptor. Accordingly, in one embodimentof the present invention, the upconversion structures include (a) PAmolecules bound to UC material and to a plasmonic metal nanoparticle,(b) a plasmonic metal nanoparticle with an UC material nanocap coveredwith PA molecules, (c) a PA-covered UC material nanoparticle withplasmonic metal nanoparticles, (d) an UC material-containingnanoparticle covered with PA molecules and a plasmonic metal nanocap,(e) a plasmonic metal nanoparticle core with an UC material nanoshellcovered with PA molecule, and (f) a PA molecule bound to UC material(attached to a plasmonics metal nanoparticle) by detachable biochemicalbond.

In the embodiment in FIGS. 12A and 12B, the bioreceptors can be antibodyprobes, DNA probes, and/or enzyme probes.

For antibody probes, antibody based targeting is more active, specificand efficient. The antibodies are selected to target a specific tumormarker (e.g., anti-epidermal growth factor receptor (EGFR) antibodiestargeted against over expressed EGFR on oral and cervical cancer cells;anti-Her2 antibodies against over expressed Her2 on breast cancer cells)Antibodies are biological molecules that exhibit very specific bindingcapabilities for specific structures. An antibody is a complexbiomolecule, made up of hundreds of individual amino acids arranged in ahighly ordered sequence. For an immune response to be produced against aparticular molecule, a certain molecular size and complexity arenecessary: proteins with molecular weights greater then 5000 Da aregenerally immunogenic. The way in which an antigen and itsantigen-specific antibody interact may be understood as analogous to alock and key fit, by which specific geometrical configurations of aunique key permits it to open a lock. In the same way, anantigen-specific antibody “fits” its unique antigen in a highly specificmanner. This unique property of antibodies is the key to theirusefulness in immunosensors where only the specific analyte of interest,the antigen, fits into the antibody binding site.

For DNA probes, the operation of gene probes is based on hybridizationprocesses Hybridization involves the joining of a single strand ofnucleic acid with a complementary probe sequence. Hybridization of anucleic acid probe to DNA biotargets (e.g., gene sequences of amutation, etc) offers an accurate measure for identifying DNA sequencescomplementary to that of the probe. Nucleic acids strands tend to bepaired to their complements in the corresponding double-strandedstructure. Therefore, a single-stranded DNA molecule will seek out itscomplement in a complex mixture of DNA containing large numbers of othernucleic acid molecules. Hence, nucleic acid probe (i.e., gene probe)detection methods are very specific to DNA sequences. Factors affectingthe hybridization or reassociation of two complementary DNA strandsinclude temperature, contact time, salt concentration, and the degree ofmismatch between the base pairs, and the length and concentration of thetarget and probe sequences.

Biologically active DNA probes can be directly or indirectly immobilizedonto a drug system, such as the EEC system (e.g., gold nanoparticle, asemiconductor, quantum dot, a glass/quartz nanoparticles, etc.), surfaceto ensure optimal contact and maximum binding. When immobilized ontogold nanoparticles, the gene probes are stabilized and, therefore, canbe reused repetitively. Several methods can be used to bind DNA todifferent supports. The method commonly used for binding DNA to glassinvolves silanization of the glass surface followed by activation withcarbodiimide or glutaraldehyde. In one embodiment, silanization methodsare used for binding to glass surfaces using 3glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane(APTS) and may be used to covalently link DNA via amino linkersincorporated either at the 3′ or 5′ end of the molecule during DNAsynthesis.

For enzyme probes, enzymes are often chosen as bioreceptors based ontheir specific binding capabilities as well as their catalytic activity.In biocatalytic recognition mechanisms, the detection is amplified by areaction catalyzed by macromolecules called biocatalysts. With theexception of a small group of catalytic ribonucleic acid molecules, allenzymes are proteins. Some enzymes require no chemical groups other thantheir amino acid residues for activity. Others require an additionalchemical component called a cofactor, which may be either one or moreinorganic ions, such as Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, or a more complexorganic or metalloorganic molecule called a coenzyme. The catalyticactivity provided by enzymes allows for much lower limits of detectionthan would be obtained with common binding techniques. The catalyticactivity of enzymes depends upon the integrity of their native proteinconformation. If an enzyme is denatured, dissociated into its subunits,or broken down into its component amino acids, its catalytic activity isdestroyed. Enzyme-coupled receptors can also be used to modify therecognition mechanisms.

The novel materials and upconverter structures of the invention includein various embodiments nanoparticles of neodymium and ytterbium dopedyttrium oxide, europium and ytterbium doped yttrium oxide, and anycombination of rare earth trivalent ions doped into a neodymium oxidenanocrystal. The dual doped yttrium oxide of composition neodymium andytterbium and also the dual doped europium and ytterbium are new for theyttrium oxide host lattice, although such dual doped systems have beenshown to work in other host lattices such as YAG.

These dual doped lanthanide glasses have been shown to upconvertefficiently on bulk materials, and thereby can provide new upconverterstructures at the nanoscale. There are a number of advantages offered bythese yttrium oxide nanostructures of the invention. The small scalesynthetic methodology for creating nanoscale yttrium oxide is easier tocontrol and produce in yttrium oxide than in YAO. The host structure ofyttrium oxide scintillates (by down conversion) at a valuable emissionwavelength to excite known pharmaceutical materials as the recipients.Finally, these combinations of dopants in yttrium oxide provide newemission colors for the yttrium oxide nanocrystal in an imaging format.

In one embodiment of the invention, a dual dopant permits excitation ofeither ion in the host glass. For instance, excitation by 980 nm lightexcites an ytterbium ion, where through transfer of energy from oneexcited state of the ytterbium ion to another dopant provides amechanism for upconversion emission of light in the ultraviolet,visible, and NIR spectral regions.

Neodymium oxide is a dielectric nanostructural material that can also besynthesized by the same polyalcohol method described above with regardto yttrium oxide nanocrystal preparation. Doped neodymium oxide isexpected to also show upconversion processes. Neodymium oxide as a hoststructure possesses lower optical phonon modes than all other oxidebased materials. Lower frequency of phonon may be best suited forelectronic transfer between ions. In general, phonon modes arevibrations in a crystal lattice whose frequencies are dependent on thecrystal lattice structure and materials. Energy released by upconversion(effectively atomic emission) is transmitted through the photons. Withphotons, energy can be transferred via Forster, Dexter, or photoncapture pathways. Meanwhile, for holes and electrons, charge tunnelingis one mechanism for energy transfer. For photons, lower phonon modestypically exhibit less destructive interference, thereby being moresuitable for upconverted emission. Accordingly, in one embodiment of theinvention, the lower energy phonon modes for neodymium oxide areexpected to provide for a stronger electron phonon coupling transfer tooccur between the dopants inside of the neodymium oxide. Neodymium oxidehas also shown the same low toxic effects as yttrium oxide and thereforeis suitable for insertion in living biological tissue.

Accordingly, the novel upconversion emitters of this invention involve anumber of configurable structures and materials which will permit theiruse in a variety of applications. Further, many of the dielectric coresdescribed in the invention exhibit down conversion properties. Theinvention in several applications described below utilizes both theupconversion and down conversion properties of a particular nanoparticlematerial system. In some of the applications described below, particlesdesigned for down conversion can be used in conjunction with separateparticles designed for upconversion.

In some embodiments of the invention, down converting materials (such asthose described herein) are used separately without the need to includeup converting materials.

Accordingly, the invention in various embodiments can use a wide varietyof down conversion materials. These down conversion materials caninclude quantum dots, semiconductor materials, alloys of semiconductormaterials, scintillation and phosphor materials, materials that exhibitX-ray excited luminescence (XEOL), organic solids, metal complexes,inorganic solids, crystals, rare earth materials (lanthanides),polymers, scintillators, phosphor materials, etc., and materials thatexhibit excitonic properties.

Further, the down conversion materials for the invention described herecan be coated with insulator materials such as for example silica whichwill reduce the likelihood of any chemical interaction between theluminescent particles and the medium. For biomedical applications,nanoparticles with toxicity as low as possible are desirable. Pure TiO₂,ZnO, and Fe₂O₃ are biocompatible. CdTe and CdSe are toxic, while ZnS,CaS, BaS, SrS and Y₂O₃ are less toxic. In addition, the toxicity ofnanoparticles can result from their inorganic stabilizers, such as TGA,or from dopants such as Eu²⁺, Cr³⁺ or Nd³⁺. Other suitable downconversion materials which would seem the most biocompatible are zincsulfide, ZnS:Mn²⁺, ferric oxide, titanium oxide, zinc oxide, zinc oxidecontaining small amounts of Al₂O₃ and AgI nanoclusters encapsulated inzeolite.

Alkali lead silicate Glass compositions were also useful fordown-converting x-rays into UV and visible. These glass compositionscontain SiO, B₂O₃, Na₂O, K₂O and PbO. The range of compositions includein mole %: SiO₂ from 44% to 73%, B₂O₃ from 0% to 3%, Na₂O from 0.5% to4%, K₂O from 0.5% to 11% and PbO from 5% to 55%. A whole range ofcompositions are possible. Furthermore, other materials can be includedto promote fluorescence including for example MgO and Ag.

In various embodiments of the invention, the following luminescentpolymers are also suitable as conversion materials: poly(phenyleneethynylene), poly(phenylene vinylene), poly(p-phenylene),poly(thiophene), poly(pyridyl vinylene), poly(pyrrole), poly(acetylene),poly(vinyl carbazole), poly(fluorenes), and the like, as well ascopolymers and/or derivatives thereof.

In the embodiment shown in FIG. 12B, the up converting agent isdisplaced from the plasmonics metal. In one embodiment, the displacementdistance effects (and in certain situations enhances) the interaction ofthe incident radiation with the up converting material. FIG. 12B-1 showsan example of the enhancement (f) of emission as a function ofwavelength for a configuration similar to that in FIG. 12B where theenergy converting molecule is displaced from the metal shell, and wherethe outer radius of the metal shell is set arbitrarily to 50 nm and b isthe value of the inner radius of the shell in units of nanometers.

In concept, the same effect occurs if the molecule were located atdifferent positions inside a metallic shell. The enhancement results areshown in FIG. 12B-2, where the outer radius of the metal shell is setarbitrarily to 50 nm and b is the value of the inner radius of the shellin units of nanometers.

As shown in FIG. 12A, the up converting agent can be disposed inside aplasmonics metal shell. The maximum enhancement effect will generallyoccur near a plasmon resonance of the metallic shell, and therefore theenhancement will generally have a strong dependence on wavelength. FIG.12B-3 shows an example of the dependence of excitation enhancement onwavelength for a configuration similar to that in FIG. 12A where theenergy converting material is covered with a plasmonics layer, where theouter radius of the shell is set arbitrarily to 50 and b is the value ofthe inner radius of the shell in units of nanometers.

Once the up converting or down converting molecule is excited by theincident radiation field, its energy is released by emission such as forexample fluorescence or phosphorescence. If for the purpose ofillustrating this embodiment of the invention, one assumes an intrinsicquantum efficiency of the molecule equal to one, the molecule, in theabsence of the shell, will radiate away all its energy due to theexciting field. In the presence of the shell, some of the radiativepower will be absorbed the shell. FIG. 12B-4 shows the dependence of theradiation (i.e., emission) on wavelength for the structure andexcitation shown in FIG. 12B-3.

FIG. 12B-5 shows the data of FIG. 12B-4 simplified to show the totalenhancement verses the inside diameter of the metal shell.

From the results discussed above, when the molecule is outside theshell, the local field is the incident radiation field plus the fieldscattered from the shell. When the molecule resides within the shellcore, the local field is the radiation field that penetrates the shell.Both the excitation of the molecule and its radiative emission arestrongly influenced by plasmon resonances excited within the shell. Inone embodiment of the invention, the enhancement for an externalmolecule is larger for a shell than for a solid sphere of the samediameter.

Both the excitation and the quantum efficiency are larger for a shellthan for a solid sphere, although both of these quantities appear topeak at different shell thicknesses. In one embodiment of the invention,the overall enhancement can range as high as 30 for a molecule outsidethe shell and about 15 for a molecule inside the shell core. In thelatter case, however, the enhancement is inhibited by a thick shell andachieves a peak value for a relatively thin shell. Two factors affectthe drop off in the enhancement as the shell becomes increasingly thin.The first is the weakening of the plasmon resonance as the volume ofmetal is reduced and the second is the further damping of the resonancedue to electron scattering within a thin shell.

Recent work by Schietinger et al. (Nano Lett. 2010, 10, 134-138)demonstrates similar plasmon enhancement of the emission of upconvertingnanocrystals. In this work, the group prepared NaYF₄ nanocrystals dopedwith Er and Yb and codeposited 5 nm Au nanoparticles to a thin film. AFMtip manipulation coupled with single particle emission spectroscopyconfirmed a 2.7 to 4.8 fold enhancement of the upconverting nanocrystalsin a thin film deposition.

The doped yttrium oxide materials described above as well as the othernanocrystalline materials of the invention are upconverters which offeran alternative to more conventional types of techniques for imaging orphoto-induced treatment. In some of the cross referenced related patentapplications, high energy photons such as X-ray or high energy particleswere used in a down conversion manner to generate subsequent ultravioletlight for interaction with drugs introduced to the body or (in otherwork) for the production of a singlet oxygen in the body or fordiagnostics via imaging the secondarily emitted light. In some of thecross referenced related patent applications, high energy photons suchas X-ray or high energy particles were used in a down conversion mannerto generate secondarily emitted light which activated an agent in themedium. The interaction of X-ray with nanoparticles and the resultantemission is thus a determining event in the down conversion process ofthe present invention. It has been discovered that the resultant lightemission for Y₂O₃ particles show at least in the range from 120 kV to320 kV an unexpected increase in emission intensity with decreasingX-ray energy.

In one embodiment of this invention, a more benign radiation source(than X-ray) that of a NIR source can be used. NIR sources are readilyavailable with commercial laser sources that operate, for example at 980and 808 nm. There are many commercially available NIR diode laser lines;these include 785, 830, 852, 915, 940, 1064, 1310, and 1550 nm inaddition to 808 and 980, which depending on the nanoscale agent andapplication, many of these are suitable for use.

The doped yttrium oxide materials described above as well as the othernanocrystalline materials of the invention are upconverters which offeran alternative to more conventional types of techniques for imaging orphoto-induced treatment. In some of the cross referenced related patentapplications, high energy photons such as X-ray or high energy particleswere used in a down conversion manner to generate subsequent ultravioletlight for interaction with drugs introduced to the body or (in otherwork) for the production of a singlet oxygen in the body or fordiagnostics via imaging the secondarily emitted light. In some of thecross referenced related patent applications, high energy photons suchas X-ray or high energy particles were used in a down conversion mannerto generate secondarily emitted light which activated an agent in themedium. The interaction of X-ray with nanoparticles and the resultantemission is thus a determining event in the down conversion process ofthe invention. It has been observed that the resultant light emissionfor Y₂O₃ particles show at least in the range from 120 kV to 320 kV anincrease in emission intensity with decreasing X-ray energy. Otherparticles or other energy ranges may well show a different trend.

In one embodiment of this invention, a more benign radiation source,that of a NIR source, can be used. NIR sources are readily availablewith commercial laser sources that operate, for example at 980 and 808nm. There are many commercially available NIR diode laser lines; theseinclude 785, 830, 852, 915, 940, 1064, 1310, and 1550 nm in addition to808 and 980, which depending on the nanoscale agent and application,many of these are suitable for use.

PEGylated-Vectors for PEPST Probes

The synthesis of these particles was first reported by Michael Faraday,who, in 1857, described the chemical process for the production ofnanosized particles of AuO from gold chloride and sodium citrate(Faraday 1857). Initial formulations of the vector, manufactured bybinding only TNF to the particles, were less toxic than native TNF andeffective in reducing tumor burden in a murine model. Subsequent studiesrevealed that the safety of this vector was primarily due to its rapiduptake and clearance in the RES. This vector was reformulated to includemolecules of thiol-derivatized polyethylene glycol (PEG-THIOL) that werebound with molecules of TNF on the gold nanoparticles surface. The newvector, PT-cAu-TNF, avoids detection and clearance by the RES, andactively and specifically sequesters TNF within a solid tumor. Thealtered biodistribution correlated to improvements. Similarly one candesign PEGylated-Au nanoparticles-PA drug to avoid detection andclearance by the RES.

Disease-Targeted PEPST Probes

Aggregation of metal (such as silver or gold) nanoparticles(nanopsheres, nanorods, etc) is often a problem, especially withcitrate-capped gold nanospheres, cetyl trimethylammonium bromide(CTAB)-capped gold nanospheres and nanorods and nanoshells because theyhave poor stability when they are dispersed in buffer solution due tothe aggregating effect of salt ions. The biocompatibility can beimproved and nanoparticle aggregation prevented by capping thenanoparticles with polyethylene glycol (PEG) (by conjugation ofthiol-functionalized PEG with metal nanoparticles). Furthermore,PEGylated nanoparticles are preferentially accumulated into tumortissues due to the enhanced permeability and retention effect, known asthe “EPR” effect [Maedaa H, Fanga J, Inutsukaa T, Kitamoto Y (2003)Vascular permeability enhancement in solid tumor: various factors,mechanisms involved and its implications. Int Immunopharmacol 3:319-328;Pactotti G F, Auer L, Weinreich D, Gola D, Pavel N McLaughlin R ETamarkin L (2004) Colloidal gold: a novel nanoparticles vector/or tumordirected drug delivery. Drug Deliv 11:169-183]. Blood vessels in tumortissue are more “leaky” than in normal tissue, and as a result,particles, or large macromolecular species or polymeric speciespreferentially extravasate into tumor tissue. Particles and largemolecules tend to stay a longer time in tumor tissue due to thedecreased lymphatic system, whereas they are rapidly cleared out innormal tissue. This tumor targeting strategy is often referred to aspassive targeting whereas the antibody-targeting strategy is calledactive targeting.

To specifically target diseased cells, specific genes or proteinmarkers, the drug systems of the present invention can be bound to abioreceptor (e.g., antibody, synthetic molecular imprint systems, DNA,proteins, lipids, cell-surface receptors, peptides, saccharides,aptamers, etc.). Immunotargeting modalities to deliver PA agentsselectively to the diseased cells and tissue provide efficientstrategies to achieving specificity, minimizing nonspecific injury tohealthy cells, and reducing the radiation intensity used.Biofunctionalization of metal nanoparticles (e.g., gold, silver) can beperformed using commonly developed and widely used procedures. There areseveral targeting strategies that can be used in the present invention:(a) nanoparticles conjugated to antibodies that recognize biomarkersspecific to the diseased cells; (b) nanoparticles passivated by poly(ethylene) glycol (PEG), which is used to increase the biocompatibilityand biostability of nanoparticles and impart them an increased bloodretention time.

Immobilization of Biomolecules to Metal Nanoparticles

The immobilization of biomolecules (PA molecules, drugs, proteins,peptides, saccharides, enzymes, antibodies, DNA, etc.) to a solidsupport can use a wide variety of methods published in the literature.Binding can be performed through covalent bonds usually takes advantageof reactive groups such as amine (—NH₂) or sulfide (—SH) that naturallyare present or can be incorporated into the biomolecule structure.Amines can react with carboxylic acid or ester moieties in high yield toform stable amide bonds. Thiols can participate in maleimide coupling,yielding stable dialkylsulfides.

A solid support of interest is gold (or silver) nanoparticles. Themajority of immobilization schemes involving Au (Ag) surfaces utilize aprior derivatization of the surface with alkylthiols, forming stablelinkages. Alkylthiols readily form self-assembled monolayers (SAM) ontosilver surfaces in micromolar concentrations. The terminus of thealkylthiol chain can be used to bind biomolecules, or can be easilymodified to do so. The length of the alkylthiol chain has been found tobe an important parameter, keeping the biomolecules away from thesurface. Furthermore, to avoid direct, non-specific DNA adsorption ontothe surface, alkylthiols have been used to block further access to thesurface, allowing only covalent immobilization through the linker[Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-7;Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-20]

Silver surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 15°. There is also a larger thiol packing density on silver,when compared to gold [Burges, J. D.; Hawkridge, F. M. Langmuir 1997,13, 3781-6]. After SAM formation on gold/silver nanoparticles,alkylthiols can be covalently coupled to biomolecules. The majority ofsynthetic techniques for the covalent immobilization of biomoleculesutilize free amine groups of a polypeptide (enzymes, antibodies,antigens, etc) or of amino-labeled DNA strands, to react with acarboxylic acid moiety forming amide bonds. As a general rule, a moreactive intermediate (labile ester) is first formed with the carboxylicacid moiety and in a later stage reacted with the free amine, increasingthe coupling yield. Successful coupling procedures include:

Binding Procedure Using N-Hydroxysuccinimide (NHS) and its Derivatives

The coupling approach involves the esterification under mild conditionsof a carboxylic acid with a labile group, an N-hydroxysuccinimide (NHS)derivative, and further reaction with free amine groups in a polypeptide(enzymes, antibodies, antigens, etc) or amine-labeled DNA, producing astable amide [Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.;Akerman, B. Langmuir 1999, 15, 4317-20]. NHS reacts almost exclusivelywith primary amine groups. Covalent immobilization can be achieved in aslittle as 30 minutes. Since H₂O competes with —NH₂ in reactionsinvolving these very labile esters, it is important to consider thehydrolysis kinetics of the available esters used in this type ofcoupling. The derivative of NHS used in FIG. 1,O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,increases the coupling yield by utilizing a leaving group that isconverted to urea during the carboxylic acid activation, hence favorablyincreasing the negative enthalpy of the reaction.

Binding Procedure Using Maleimide

Maleimide can be used to immobilize biomolecules through available —SHmoieties (FIG. 3). Coupling schemes with maleimide have been provenuseful for the site-specific immobilization of antibodies, Fabfragments, peptides, and SH-modified DNA strands. Sample preparation forthe maleimide coupling of a protein involves the simple reduction ofdisulfide bonds between two cysteine residues with a mild reducingagent, such as dithiothreitol, 2-mercaptoethanol ortris(2-carboxyethyl)phosphine hydrochloride. However, disulfidereduction will usually lead to the protein losing its naturalconformation, and might impair enzymatic activity or antibodyrecognition. The modification of primary amine groups with2-iminothiolane hydrochloride (Traut's reagent) to introduce sulfydrylgroups is an alternative for biomolecules lacking them. Free sulfhydrylsare immobilized to the maleimide surface by an addition reaction tounsaturated carbon-carbon bonds [Jordan, C. E., et al., 1997].

Binding Procedure Using Carbodiimide.

Surfaces modified with mercaptoalkyldiols can be activated with1,1′-carbonyldiimidazole (CDI) to form a carbonylimidazole intermediate.A biomolecule with an available amine group displaces the imidazole toform a carbamate linkage to the alylthiol tethered to the surface[Potyrailo, R. A., et al., 1998].

Other Experimental Procedures to Conjugate Biomolecules to Metal (e.g.,Silver, Gold) Nanoparticles.

Nanoparticles of metal colloid hydrosols are generally prepared byrapidly mixing a solution of AgNO₃ with ice-cold NaBH. To develop a SMPprobes, one needs to bind a DNA segment to a nanoparticle of silver orgold. The immobilization of biomolecules (e.g., DNA, antibodies,enzymes, etc.) to a solid support through covalent bonds usually takesadvantage of reactive groups such as amine (—NH₂) or sulfide (—SH) thatnaturally are present or can be incorporated into the biomoleculestructure. Amines can react with carboxylic acid or ester moieties inhigh yield to form stable amide bonds. Thiols can participate inmaleimide coupling yielding stable dialkylsulfides.

In the present invention, we use silver nanoparticles. The majority ofimmobilization schemes involving Ag surfaces utilize a priorderivatization of the surface with alkylthiols, forming stable linkages.Alkylthiols readily form self-assembled monolayers (SAM) onto silversurfaces in micromolar concentrations. The terminus of the alkylthiolchain can be directly used to bind biomolecules, or can be easilymodified to do so. The length of the alkylthiol chain was found to be animportant parameter, keeping the biomolecules away from the surface.Furthermore, to avoid direct, non-specific DNA adsorption onto thesurface, alkylthiols were used to block further access to the surface,allowing only covalent immobilization through the linker.

Silver/gold surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 15°. There is also a larger thiol packing density on silver,when compared to gold.

After SAM formation on silver nanoparticles, alkylthiols can becovalently coupled to biomolecules. The majority of synthetic techniquesfor the covalent immobilization of biomolecules utilize free aminegroups of a polypeptide (enzymes, antibodies, antigens, etc) or ofamino-labeled DNA strands, to react with a carboxylic acid moietyforming amide bonds. As a general rule, a more active intermediate(labile ester) is first formed with the carboxylic acid moiety and in alater stage reacted with the free amine, increasing the coupling yield.Successful coupling procedures include:

The coupling approach used to bind DNA to a silver nanoparticle involvesthe esterification under mild conditions of a carboxylic acid with alabile group, an N-hydroxysuccinimide (NHS) derivative, and furtherreaction with free amine groups in a polypeptide (enzymes, antibodies,antigens, etc) or amine-labeled DNA, producing a stable amide [4]. NHSreacts almost exclusively with primary amine groups. Covalentimmobilization can be achieved in as little as 30 minutes. Since H₂Ocompetes with —NH₂ in reactions involving these very labile esters, itis important to consider the hydrolysis kinetics of the available estersused in this type of coupling. The derivative of NHS used,O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,increases the coupling yield by utilizing a leaving group that isconverted to urea during the carboxylic acid activation, hence favorablyincreasing the negative enthalpy of the reaction.

PEPST UC-PA Probe with Detachable PA.

For some photoactive drug requiring that the PA molecule to enter thenucleus. FIG. 13 shows an embodiment of a PEPST-UC probe where the PAdrug molecule is bound to the metal nanoparticles via a linker (FIG.13A) that can be cut by a photon radiation (FIG. 13B). Such a probe isuseful for therapy modalities where the PA molecules have to enter thenucleus, e.g., psoralen molecules need to enter the nucleus of cells andintercalate onto DNA (FIG. 13C). Since it is more difficult for metalnanoparticles to enter the cell nucleus than for smaller molecules, itis desirable to PEPST-UC probes that have releasable PA molecules.

The novel materials and upconverter structures of the invention includein various embodiments nanoparticles of neodymium and ytterbium dopedyttrium oxide, europium and ytterbium doped yttrium oxide, and anycombination of rare earth trivalent ions doped into a neodymium oxidenanocrystal. The dual doped yttrium oxide of composition neodymium andytterbium and also the dual doped europium and ytterbium are new for theyttrium oxide host lattice, although such dual doped systems have beenshown to work in other host lattices such as YAG.

These dual doped lanthanide glasses have been shown to upconvertefficiently on bulk materials, and thereby can provide new upconverterstructures at the nanoscale. There are a number of advantages offered bythese yttrium oxide nanostructures of the invention. The small scalesynthetic methodology for creating nanoscale yttrium oxide is easier tocontrol and produce in yttrium oxide than in YAG. The host structure ofyttrium oxide scintillates (by down conversion) at a valuable emissionwavelength to excite known pharmaceutical materials as the recipients.Finally, these combinations of dopents in yttrium oxide provide newemission colors for the yttrium oxide nanocrystal in an imaging format.

In one embodiment of the invention, a dual dopant permits excitation ofeither ion in the host glass. For instance, excitation by 980 nm lightexcites a ytterbium ion, where through transfer of energy from oneexcited state of the ytterbium ion to another dopant provides amechanism for upconversion emission of light in the ultraviolet,visible, and NIR spectral regions.

Neodymium oxide is a novel dielectric nanostructural material that canalso be synthesized by the same polyalcohol method described above withregard to yttrium oxide nanocrystal preparation. Doped neodymium oxideis expected to also show upconversion processes. Neodymium oxide as ahost structure possesses lower optical phonon modes than all other oxidebased materials. Lower frequency of phonon may be best suited forelectronic transfer between ions. In general, phonon modes arevibrations in a crystal lattice whose frequencies are dependent on thecrystal lattice structure and materials. Energy released by upconversion(effectively atomic emission) is transmitted through the photons. Withphotons, energy can be transferred via Forster, Dexter, or photoncapture pathways. Meanwhile, for holes and electrons, charge tunnelingis one mechanism for energy transfer. For photons, lower phonon modestypically exhibit less destructive interference, thereby being moresuitable for upconverted emission. Accordingly, in one embodiment of theinvention, the lower energy phonon modes for neodymium oxide areexpected to provide for a stronger electron phonon coupling transfer tooccur between the dopants inside of the neodymium oxide. Neodymium oxidehas also shown the same low toxic effects as yttrium oxide and thereforeis suitable for insertion in living biological tissue.

Accordingly, the novel upconversion emitters of this invention involve anumber of configurable structures and materials which will permit theiruse in a variety of applications. Further, many of the dielectric coresdescribed in the invention exhibit down conversion properties. Theinvention in several applications described below utilizes both theupconversion and down conversion properties of a particular nanoparticlematerial system. In some of the application described below, particlesdesigned for down conversion can be used in conjunction with separateparticles designed for upconversion.

Accordingly, the invention can use a wide variety of down conversionmaterials. These down conversion materials can include quantum dots,semiconductor materials, scintillation and phosphor materials, materialsthat exhibit X-ray excited luminescence (XEOL), organic solids, metalcomplexes, inorganic solids, crystals, rare earth materials(lanthanides), polymers, scintillators, phosphor materials, etc., andmaterials that exhibit excitonic properties.

Further, the down conversion materials for the invention described herecan be coated with insulator materials such as for example silica whichwill reduce the likelihood of any chemical interaction between theluminescing particles and the medium. For biological applications ofinorganic nanoparticles, one of the major limiting factors is theirtoxicity. Generally speaking, all semiconductor nanoparticles are moreor less toxic. For biomedical applications, nanoparticles with toxicityas low as possible are desirable or else the nanoparticles have toremain separated from the medium. Pure TiO₂, ZnO, and Fe₂O₃ arebiocompatible. CdTe and CdSe are toxic, while ZnS, CaS, BaS, SrS andY₂O₃ are less toxic. In addition, the toxicity of nanoparticles canresult from their inorganic stabilizers, such as TGA, or from dopantssuch as Eu²⁺, Cr³⁺ or Nd³⁺. Other suitable down conversion materialswhich would seem the most biocompatible are zinc sulfide, ZnS:Mn²⁴,ferric oxide, titanium oxide, zinc oxide, zinc oxide containing smallamounts of Al₂O₃ and AgI nanoclusters encapsulated in zeolite. Fornon-medical applications, where toxicity may not be as critical aconcern, the following materials (as well as those listed elsewhere) areconsidered suitable: lanthanum and gadolinium oxyhalides activated withthulium; Er³⁺ doped BaTiO₃ nanoparticles, Yb⁺ doped CsMnCl₃ and RbMnCl₃,BaFBr:Eu²⁺ nanoparticles, Cesium Iodine, Bismuth Germanate, CadmiumTungstate, and CsBr doped with divalent Eu.

In various embodiments of the invention, the following luminescentpolymers are also suitable as conversion materials: poly(phenyleneethynylene), poly(phenylene vinylene), poly(p-phenylene),poly(thiophene), poly(pyridyl vinylene), poly(pyrrole), poly(acetylene),poly(vinyl carbazole), poly(fluorenes), and the like, as well ascopolymers and/or derivatives thereof.

The doped yttrium oxide materials described above as well as the othernanocrystalline materials of the invention are upconverters which offeran alternative to more conventional types of techniques for imaging orphoto-induced treatment. In the cross referenced related patentapplications and in other work, high energy photons such as X-ray orhigh energy particles were used in a down conversion manner to generatesubsequent ultraviolet light for interaction with drugs introduced tothe body or for the production of a singlet oxygen in the body or fordiagnostics via imaging the secondarily emitted light. In thisinvention, a more benign radiation source that of a NIR source can beused. NIR sources are readily available with commercial laser sourcesthat operate, for example at 980 and 808 nm. There are many commerciallyavailable NIR diode laser lines; these include 785, 830, 852, 915, 940,1064, 1310, and 1550 nm in addition to 808 and 980, which depending onthe nanoscale agent and application, many of these are suitable for use.

These IR frequencies have significant penetration into the human bodyand permit the primary excitation λ₁ to penetrate subcutaneously intothe body tissue. Upon their penetration into the body tissue, thedielectric core of the invention interacts with the incident radiationλ₁ to generate the secondary light λ₂ as described above. Therefore,permitting the generation in situ to the body of a wavelength λ₂ whichmay be in the UV or visible range is appropriate for activations ofpsoralen or other types of drugs known to be activated by a UV orvisible light source.

Since the dielectric cores of this invention have the ability to beselectively stimulated by discrete wavelengths of λ₁ and producediscrete emission wavelengths at λ₂, the medial applications can bemanipulated so that a number of dual purpose diagnostic/treatment toolscan be produced.

For example, in one embodiment of the invention, a material such as theabove-described co-doped yttrium oxide is introduced into the body.Yttrium oxide as a host is known to be a down converter from X-rayradiation. In this particular example, X-ray incident radiation on theyttrium oxide will produce UV light which would in turn be used toactivate drugs such as psoralen for the treatment of cancer.

Meanwhile, the co-doped yttrium oxide as a upconverter could be usedwhere the NIR excitation could produce an emission at a wavelength thatwas different than that produced from the X-ray down conversionradiation. In this manner, the progression of the yttrium oxide (withdrug attached as the recipient 4) into a target organ to be treatedcould be monitored using the NIR light as the excitation source andcollecting the visible light in some type of CCD camera. Once theyttrium oxide particles were absorbed into the respective tumor cellsfor treatment, at that point in time, X-ray radiation could be initiatedand thereby activating the psoralen tagged yttrium oxide and providingan effective means for treating the tumor cell.

Alternatively, in another dual purpose diagnostic/treatment example, onecan choose a system where the NIR wavelength is specifically tuned fordiagnostics as explained above while excitation with a separatewavelength of NIR can be used to produce UV light (through anotherupconversion channel) that would itself activate a recipient molecule(e.g. psoralen for cancer treatment) without the necessity of X-ray anddown conversion activation. This feature then permits one to use a drugwhich either would be acceptable for deep body penetration through X-rayradiation or would be acceptable for more shallow body penetrationthrough NIR radiation to treat cancer cells that were located indifferent parts of the body relative to the surface of the body.Moreover, fiber optics could be used to direct the NIR light (through asurgical incision for example) directly to a target. By locallyactivating the psoralen and by the known autovaccine effect, thisinitially local NIR activated treatment may be effective at treatingcancer outside the NIR irradiated area.

Examples of such dual use drugs which all exhibit NIR activation andupconversion for the purpose of imaging and/or to excite psoralen wouldinclude the dual dopants of yttrium oxide, the dual dopants of neodymiumoxide, triply doped ytterbium thulium neodymium oxides, the dual dopantsof sodium yttrium fluoride, and the dual dopants of lanthanum fluoride.For example, by providing a ytterbium-thulium doped yttrium oxidecontaining 95% verses 5% dopant concentration with another lanthanide,one will produce diagnostic/treatment functions through pure NIRexcitation, having the drug treatment excitable at 980 nanometers versesthe diagnostic imaging process excitable at 808 nanometers withdifferent emissions coming from each excitation process.

Nanoparticle Chain for Dual Plasmonics Effect

As discussed previously, there is the need to develop nanoparticlesystems that can have dual (or multi) plasmonics resonance modes. FIG.14 illustrates an embodiment of the present invention PEPST probe havinga chain of metal particles having different sizes and coupled to eachother, which could exhibit such dual plasmonics-based enhancement. Forexample the parameters (size, metal type, structure, etc) of the largernanoparticle (FIG. 14, left) can be tuned to NIR, VIS or UV light whilethe smaller particle (FIG. 14, right) can be tuned to X ray. There isalso a coupling effect between these particles.

These nanoparticle chains are useful in providing plasmonics enhancementof both the incident radiation used (for example, x-ray activation ofCdS) as well as plasmonics enhancement of the emitted radiation thatwill then activate the PA. Similar nanoparticles systems have been usedas nanolens [Self-Similar Chain of Metal Nanospheres as an EfficientNanolens, Kuiru Li, Mark I. Stockman, and David J. Bergman, PhysicalReview Letter, VOLUME 91, NUMBER 22, 227402-1, 2003].

Drug Delivery Platforms

Liposome Delivery of Energy Modulation Agent-PA Systems

The field of particle-based drug delivery is currently focused on twochemically distinct colloidal particles, liposomes and biodegradablepolymers. Both delivery systems encapsulate the active drug. The drug isreleased from the particle as it lyses, in the case of lipsomes, ordisintegrates, as described for biodegradable polymers. One embodimentof the present invention uses liposomal delivery of energy modulationagent-PA systems (e.g., gold nanoshells) for therapy. An exemplaryembodiment is described below, but is not intended to be limiting to thespecific lipids, nanoparticles or other components recited, but ismerely for exemplary purposes:

Preparation of Liposomes.

The liposome preparation method is adapted from Hölig et. al Hölig, P.,Bach, M., Völkel, T., Nahde, T., Hoffmann, S., Müller, R., andKontermann, R. E., Novel RGD lipopeptides for the targeting of liposomesto integrin-expressing endothelial and melanoma cells. ProteinEngineering Design and Selection, 2004. 17(5): p. 433-441]. Briefly, thelipids PEG-DPPE, PC, and Rh-DPPE are mixed in chloroform in a roundbottom flask and evaporated (Hieroglyph Rotary Evaporator, RoseScientific Ltd., Edmonton, Alberta, Canada) to eliminate chloroform. Thedry film is dehydrated into aqueous phase with using PBS solution. A drylipid film is prepared by rotary evaporation from a mixture of PC,cholesterol, and PEG-DPPE and then hydrated into aqueous phase usingPBS. The mixture is vigorously mixed by overtaxing and bath solicited(Instrument, Company) and the suspension extruded through polycarbonatefilter using Liposofast apparatus (Avestin Inc., Ottawa, ON, Canada)(pore-size 0.8 μm). Preparation of liposomes is performed as follows;0.1 mmol of PC is dispersed in 8 ml of chloroform and supplemented with0.5 mol of PEG-DPPE in 20 ml of chloroform. 0.3 mmol rhodamine-labeledphosphatidylethanolamine (Rh-DPPE) is then incorporated into theliposomes. The organic solvents are then removed by rotary evaporationat 35° C. for 2 h leaving a dry lipid film. Gold nanoshells areencapsulated into liposomes by adding them to the PBS hydration bufferand successively into the dry lipid film. This mixture is emulsified ina temperature controlled sonicator for 30 minutes at 35° C. followed byvortexing for 5 min. Encapsulated gold nanoshells are separated fromunencapsulated gold nanoshells by gentle centrifugation for 5 minutes at2400 r.p.m (1200 g). The resulting multilamellar vesicles suspension isextruded through polycarbonate filter using Liposofast apparatus(Avestin Inc., Ottawa, ON, Canada) (pore-size 0.8 μm). The aqueousmixture is obtained and stored at 4° C.

Fabrication of Gold Nanoparticles:

The Frens method [Frens, G., Controlled nucleation for the regulation ofthe particle size in monodisperse gold solutions. Nature (London) PhysSci, 1973. 241; p. 20-22] can be used in the present invention tosynthesize a solution of gold nanoparticles ranging in diameter from8-10 nm. Briefly, 5.0×10⁻⁶ mol of HAuCl₄ is dissolved in 19 ml ofdeionized water producing a faint yellowish solution. This solution isheated with vigorous stirring in a rotary evaporator for 45 minutes. 1ml of 0.5% sodium citrate solution is added and the solution is stirredfor an additional 30 minutes. The color of the solution graduallychanged from the initial faint yellowish to clear, grey, purple andfinally a tantalizing wine-red color similar to merlot. The sodiumcitrate used serves in a dual capacity, first acting as a reducingagent, and second, producing negative citrate ions that are adsorbedonto the gold nanoparticles introducing surface charge that repels theparticles and preventing nanocluster formation.

Preparation and Internalization of Liposome-Encapsulated GoldNanoshells:

Liposome-encapsulated gold nanoshells are incubated with MCF-7 cellsgrown on partitioned cover-slips for intracellular delivery. This isdone by adding 10 pd of liposome-encapsulated gold nanoshells per 1 mlof cell culture medium. This is incubated for 30 minutes in a humidified(86% RH) incubator at 37° C. and 5% CO₂. This cell is used forlocalization studies; to track the rhodamine-DPPE-labeled liposomes intothe cytoplasm of the MCF-7 cell. After incubation, the cells grown oncover-slips are washed three times in cold PBS and fixed using 3.7%formaldehyde in PBS. Rhodamine staining by rhodamine-DPPE-labeledliposomes is analyzed using a Nikon Diaphot 300 inverted microscope(Nikon, Inc., Melville, N.Y.).

Non-Invasive Cleavage of the Drug System In Vivo

After delivery of the drug system into the cell, there is sometimes theneed to have the PA system (e.g. psoralen) in the nucleus in order tointeract with DNA. If the PA is still linked to the energy modulationagent, both of them have to be transported into the nucleus. In the casewith gold nanoparticles as the energy modulation agent system, there areseveral methods to incubate cells in vitro. For in vivo applications,one can link the PA to the gold nanoparticles using a chemical linkagethat can be released (or cut) using non-invasive methods such asinfrared, microwave, or ultrasound waves. An example of linkage isthrough a chemical bond or through a bioreceptor, such as an antibody.In this case, the PA is the antigen molecule bound to the energymodulation agent system that has an antibody targeted to the PA.

When the energy modulation agent-Ab-PA enters the cell, the PA moleculescan be released from the energy modulation agent Ab system. To releasethe PA molecule from the antibody, chemical reagents can be used tocleave the binding between antibody and antigen, thus regenerating thebiosensor [Vo-Dinh et al, 1988]. This chemical procedure is simple butis not practical inside a cell due to possible denaturation of the cellby the chemical. In previous studies, it has been demonstrated that thegentle but effective MHz-range ultrasound has the capability to releaseantigen molecules from the antibody-energy modulation agent system[Moreno-Bondi, M., Mobley, J., and Vo-Dinh, T., “RegenerableAntibody-based Biosensor for Breast Cancer,” J. Biomedical Optics, 5,350-354 (2000)]. Thus, an alternative embodiment is to use gentleultrasonic radiation (non-invasively) to remove the PA (antigen) fromthe antibody at the energy modulation agent system.

In a preferred embodiment, the PA molecule is bound to the energymodulation agent by a chemically labile bond [Jon A. Wolff, and David B.Rozema, Breaking the Bonds: Non-viral Vectors Become Chemically Dynamic,Molecular Therapy (2007) 16(1), 8-15]. A promising method of improvingthe efficacy of this approach is to create synthetic vehicles (SVs) thatare chemically dynamic, so that delivery is enabled by the cleavage ofchemical bonds upon exposure to various physiological environments orexternal stimuli. An example of this approach is the use of maskedendosomolytic agents (MEAs) that improve the release of nucleic acidsfrom endosomes, a key step during transport. When the MEA enters theacidic environment of the endosome, a pH-labile bond is broken,releasing the agent's endosomolytic capability.

Use of Ferritin and Apoferritin as Targeted Drug Delivery

Another embodiment to deliver the energy modulation agent-PA drugsinvolves the use of ferritin and apoferritin compounds. There isincreasing interest in ligand-receptor-mediated delivery systems due totheir non-immunogenic and site-specific targeting potential to theligand-specific bio-sites. Platinum anticancer drug have beenencapsulated in apoferritin [Zhen Yang, Xiaoyong Wang, Huajia Diao,Junfeng Zhang, Hongyan Li, Hongzhe Sun and Zijian Guo, Encapsulation ofplatinum anticancer drugs by apoferritin, Chem. Commun. 33, 2007,3453-3455]. Ferritin, the principal iron storage molecule in a widevariety of organisms, can also be used as a vehicle for targeted drugdelivery. It contains a hollow protein shell, apoferritin, which cancontain up to its own weight of hydrous ferric oxide-phosphate as amicrocrystalline micelle. The 24 subunits of ferritin assembleautomatically to form a hollow protein cage with internal and externaldiameters of 8 and 12 nm, respectively. Eight hydrophilic channels ofabout 0.4 nm, formed at the intersections of subunits, penetrate theprotein shell and lead to the protein cavity. A variety of species suchas gadolinium (Gd³⁺) contrast agents, desferrioxamine B, metal ions, andnanoparticles of iron salts can be accommodated in the cage ofapoferritin. Various metals such as iron, nickel, chromium and othermaterials have been incorporated into apoferritin [Iron incorporationinto apoferritin. The role of apoferritin as a ferroxidase, The Journalof Biological Chemistry [0021-9258] Bakker yr: 1986 vol: 261 iss: 28 pg:13182-5; Mitsuhiro Okuda¹, Kenji Iwahori², Ichiro Yamashita², HideyukiYoshimura¹*, Fabrication of nickel and chromium nanoparticles using theprotein cage of apoferritin, Biotechnology Bioengineering, Volume 84,Issue 2, Pages 187-194]. Zinc selenide nanoparticles (ZnSe NPs) weresynthesized in the cavity of the cage-shaped protein apoferritin bydesigning a slow chemical reaction system, which employs tetraaminezincion and selenourea. The chemical synthesis of ZnSe NPs was realized in aspatially selective manner from an aqueous solution, and ZnSe cores wereformed in almost all apoferritin cavities with little bulk precipitation[Kenji Iwahori, Keiko Yoshizawa, Masahiro Muraoka, and Ichiro Yamashita,Fabrication of ZnSe Nanoparticles in the Apoferritin Cavity by Designinga Slow Chemical Reaction System, Inorg. Chem., 44 (18), 6393-6400,2005].

A simple method for synthesizing gold nanoparticles stabilized by horsespleen apoferritin (HSAF) is reported using NaBH₄ or3-(N-morpholino)propanesulfonic acid (MOPS) as the reducing agent [LeiZhang, Joe Swift, Christopher A. Butts, Vijay Yerubandi and Ivan J.Dmochowski, Structure and activity of apoferritin-stabilized goldnanoparticles, Journal of Inorganic Biochemistry, Vol. 101, 1719-1729,2007]. Gold sulfite (AuaS) nanoparticles were prepared in the cavity ofthe cage-shaped protein, apoferritin. Apoferritin has a cavity, 7 nm indiameter, and the diameter of fabricated Au₂S nanoparticles is about thesame size with the cavity and size dispersion was small. [KeikoYoshizawa, Kenji Iwahori, Kenji Sugimoto and Ichiro Yamashita,Fabrication of Gold Sulfide Nanoparticles Using the Protein Cage ofApoferritin, Chemistry Letters, Vol. 35 (2006), No. 10 p. 1192]. Thus,in a preferred embodiment, the PA or energy modulation agent-PAcompounds are encapsulated inside the apoferrtin shells.

Use of Ferritin and Apoferritin as Enhanced Targeting Agents

It was reported that ferritin could be internalized by some tumortissues, and the internalization was associated with themembrane-specific receptors [S. Fargion, P. Arosio, A. L. Fracanzoni, V.Cislaghi, S. Levi, A. Cozzi, A Piperno and A. G. Firelli, Blood, 1988,71, 753-757; P. C. Adams, L. W. Powell and J. W. Halliday, Hepatology,1988, 8, 719-721]. Previous studies have shown that ferritin-bindingsites and the endocytosis of ferritin have been identified in neoplasticcells [M. S. Bretscher and J. N. Thomson, EMBO J., 1983, 2, 599-603].Ferritin receptors have the potential for use in the delivery ofanticancer drugs into the brain [S. W. Hulet, S. Powers and J. R.Connor, J. Neurol. Sci., 1999, 165, 48-55]. In one embodiment, thepresent invention uses ferritin or apoferritin to both encapsulate PAand energy modulation agent-PA systems and also target tumor cellsselectively for enhanced drug delivery and subsequent phototherapy. Inthis case no additional bioreactors are needed.

FIG. 15 schematically illustrates the use of encapsulated photoactiveagents (FIG. 15A) for delivery into tissue and subsequent release of thephotoactive drugs after the encapsulated systems enter the cell. Notethe encapsulated system can have a bioreceptor for selective tumortargeting (FIG. 15B). Once inside the cell, the capsule shell (e.g.,liposomes, apoferritin, etc.) can be broken (FIG. 22C) usingnon-invasive excitation (e.g., ultrasound, RF, microwave, IR, etc) inorder to release the photoactive molecules that can get into the nucleusand bind to DNA (FIG. 22D).

Non-Invasive Phototherapy Using PEPST Modality

FIG. 16 illustrates the basic operating principle of the PEPST modality.The PEPST photoactive drug molecules are given to a patient by oralingestion, skin application, or by intravenous injection. The PEPSTdrugs travel through the blood stream inside the body towards thetargeted tumor (either via passive or active targeting strategies). Ifthe disease is systematic in nature a photon radiation at suitablewavelengths is used to irradiate the skin of the patient, the lightbeing selected to penetrate deep inside tissue (e.g., NIR or X ray). Forsolid tumors, the radiation light source is directed at the tumor.Subsequently a treatment procedure can be initiated using delivery ofenergy into the tumor site. One or several light sources may be used asdescribed in the previous sections. One embodiment of therapy comprisessending NIR radiation using an NIR laser through focusing optics.Focused beams of other radiation types, including but not limited to Xray, microwave, radio waves, etc. can also be used and will depend uponthe treatment modalities used.

The present invention treatment may also be used for inducing an autovaccine effect for malignant cells, including those in solid tumors. Tothe extent that any rapidly dividing cells or stem cells may be damagedby a systemic treatment, then it may be preferable to direct thestimulating energy directly toward the tumor, preventing damage to mostnormal, healthy cells or stem cells by avoiding photoactivation orresonant energy transfer of the photoactivatable agent

Exciton-Plasmon Enhanced Phototherapy (EPEP)

Basic Principle of Exciton-Induced Phototherapy

Excitons in Solid Materials

Excitons are often defined as “quasiparticles” inside a solid material.In solid materials, such as semiconductors, molecular crystals andconjugated organic materials, light excitation at suitable wavelength(such as X ray, UV and visible radiation, etc) can excite electrons fromthe valence band to the conduction band. Through the Coulombinteraction, this newly formed conduction electron is attracted, to thepositively charged hole it left behind in the valence band. As a result,the electron and hole together form a bound state called an exciton.(Note that this neutral bound complex is a “quasiparticle” that canbehave as a boson—a particle with integer spin which obeys Bose-Einsteinstatistics; when the temperature of a boson gas drops below a certainvalue, a large number of bosons ‘condense’ into a single quantumstate—this is a Bose-Einstein condensate (BEC). Exciton production isinvolved in X-ray excitation of a solid material. Wide band-gapmaterials are often employed for transformation of the x-ray toultraviolet/visible photons in the fabrication of scintillators andphosphors [Martin Nikl, Scintillation detectors for x-rays, Meas. Sci.Technol. 17 (2006) R37-R54]. The theory of excitons is well known inmaterials research and in the fabrication and applications ofsemiconductors and other materials. However, to the present inventors'knowledge, the use of excitons and the design of energy modulation agentmaterials based on exciton tunability for phototherapy have not beenreported.

During the initial conversion a multi-step interaction of a high-energyX-ray photon with the lattice of the scintillator material occursthrough the photoelectric effect and Compton scattering effect; forX-ray excitation below 100 keV photon energy the photoelectric effect isthe main process. Many excitons (i.e., electron-hole pairs) are producedand thermally distributed in the conduction bands (electrons) andvalence bands (holes). This first process occurs within less than 1 ps.In the subsequent transport process, the excitons migrate through thematerial where repeated trapping at defects may occur, leading to energylosses due to nonradiative recombination, etc. The final stage,luminescence, consists in consecutive trapping of the electron-holepairs at the luminescent centers and their radiative recombination. Theelectron-hole pairs can be trapped at the defects and recombine,producing luminescent. Luminescent dopants can also be used as traps forexciton.

Exciton Traps

Exciton traps can be produced using impurities in the crystal hostmatrix. In impure crystals with dipolar guest molecules the electrontrap states may arise when electron is localized on a neighbor of theimpurity molecule. Such traps have been observed in anthracene dopedwith carbazole [Kadshchuk, A. K., Ostapenko, N. I., Skryshevskii, Yu.A., Sugakov, V. I. and Susokolova, T. O., Mol. Cryst. and Liq. Cryst.,201, 167 (1991)]. The formation of these traps is due to the interactionof the dipole moment of the impurity with charge carrier. When theconcentration of the dopant (or impurities) is increased, spectraexhibit additional structure of spectrum due to the trapping of carrierson clusters of impurity molecules. Sometimes, impurities and dopants arenot required: the electron or exciton can also be trapped on astructural defect in such crystals due to the electrostatic interactionwith reoriented dipole moment of disturbed crystal molecules [S. V.Izvekov, V. I. Sugakov, Exciton and Electron Traps on Structural Defectsin Molecular Crystals with Dipolar Molecules, Physica Scripta. Vol. T66,255-257, 1996]. One can design structural defects in molecular crystalsthat serve as exiton traps. The development of GaAs/AlGaAsnanostructures and use of nanofabrication technologies can designengineered exciton traps with novel quantum mechanical properties inmaterials

Design, Fabrication and Operation of EIP Probes

FIG. 17 shows various embodiments of EIP probes that can be designed:

-   -   (A) probe comprising PA molecules bound (through a linker, which        can be fixed or detachable) to an energy modulation agent        particle that can produce excitons under radiative excitation at        a suitable wavelength (e.g., X-ray). In this preferred        embodiment, the energy modulation agent materials have        structural defects that serve as traps for excitons.    -   (B) probe comprising PA molecules bound (through a linker, which        can be fixed or detachable) to an energy modulation agent        particle that can produce excitons under radiative excitation at        a suitable wavelength (e.g., X-ray). In this preferred        embodiment, the energy modulation agent materials have        impurities or dopant molecules that serve as traps for excitons.

EIP Probes with Tunable Emission:

The embodiment in probes B provide the capability to tune the energyconversion from an X ray excitation source into a wavelength of interestto excite the PA molecules. In 1976, D'Silva et al demonstrated thatpolynuclear aromatic hydrocarbons (PAH) molecules doped in a frozenn-alkane solids could be excited by X-ray and produce luminescence atvisible wavelengths characteristics of their luminescence spectra. [A.P. D'Silva, G. J. Oestreich, and V. A. Fassel, X-ray excited opticalluminescence of polynuclear aromatic hydrocarbons, Anal. Chem.; 1976;48(6) pp 915-917]. Tunable EIP probes can be designed to contain suchluminescent dopants such as highly luminescent PAHs exhibitingluminescence emission in the range of 300-400 nm suitable to activatepsoralen. A preferred embodiment of the EIP with tunable emissioncomprises a solid matrix (semiconductors, glass, quartz, conjugatedpolymers, etc) doped with naphthalene, phenanthrene, pyrene or othercompounds exhibiting luminescence (fluorescence) in the 300-400 nm range[T. Vo-Dinh, Multicomponent analysis by synchronous luminescencespectrometry, Anal. Chem.; 1978; 50(3) pp 396-401]. The EEC matrix couldbe a semiconductor material, preferably transparent at opticalwavelength of interest (excitation and emission).

Other dopant species such as rare earth materials can also be used asdopants. FIG. 27 shows the X ray excitation optical luminescence (XEOL)of Europium doped in a matrix of BaFBr, emitting at 370-420 nm. U.S.Patent Application Publication No. 2007/0063154 (hereby incorporated byreference) describes these and other nanocomposite materials (andmethods of making them) suitable for XEOL.

Various EIP probes can be designed:

(A) A probe comprising PA molecules bound around the energy modulationagent particle or embedded in a shell around an energy modulation agentparticle that can produce excitons under radiative excitation at asuitable wavelength (e.g., X-ray). In this preferred embodiment, theenergy modulation agent materials has structural defects that serve astraps for excitons.

(B) A probe comprising PA molecules bound around the energy modulationagent particle or embedded in a shell around an energy modulation agentparticle that can produce excitons under radiative excitation at asuitable wavelength (e.g., X-ray). In this preferred embodiment, theenergy modulation agent materials have impurities or dopant moleculesthat serve as traps for excitons.

Principle of Exciton-Plasmon Enhanced Phototherapy (EPEP)

There is recent interest in an advanced photophysical concept involvingquantum optical coupling between electronic states (excitons), photonsand enhanced electromagnetic fields (plasmons). Such a concept involvingcoupling between excitons and plasmons can be used to enhance aphototherapy modality, referred to as Exciton-Plasmon EnhancedPhototherapy (EPEP).

A fundamental key concept in photophysics is the formation of newquasiparticles from admixtures of strongly-coupled states. Such mixedstates can have unusual properties possessed by neither originalparticle. The coupling between excitons and plasmons can be either weakor strong. When the light-matter interaction cannot be considered as aperturbation, the system is in the strong coupling regime. Bellesa et alshowed a strong coupling between a surface plasmon (SP) mode and organicexcitons occurs; the organic semiconductor used is a concentratedcyanine dye in a polymer matrix deposited on a silver film [Ref: J.Bellessa, * C. Bonnand and J. C. Plenet, J. Mugnier, Strong Couplingbetween Surface Plasmons and Excitons in an Organic Semiconductor, Phys.Rev. Lett, 93 (3), 036404-1, 2004]. Govorov et al describe thephotophysical properties of excitons in hybrid complexes consisting ofsemiconductor and metal nanoparticles. The interaction betweenindividual nanoparticles can produce an enhancement or suppression ofemission. Enhanced emission comes from electric field amplified by theplasmon resonance, whereas emission suppression is a result of energytransfer from semiconductor to metal nanoparticles. [Alexander O.Govorov, *,† Garnett W Bryant,‡ Wei Zhang,† Timur Skeini,† Jaebeom Lee,§Nicholas A. Kotov,§ Joseph M. Slocik,| and Rajesh R Naik|,Exciton-Plasmon Interaction and Hybrid Excitons in Semiconductor-MetalNanoparticle Assemblies, Nano Lett., Vol. 6, No. 5, 984, 2006]. Bondarevet al also described a theory for the interactions between excitonicstates and surface electromagnetic modes in small-diameter (<1 nm)semiconducting single-walled carbon nanotubes (CNs). [I. V. Bondarev, KTatur and L. M. Woods, Strong exciton-plasmon coupling in semiconductingcarbon nanotubes].

Fedutik et al reported about the synthesis and optical properties of acomposite metal-insulator-semiconductor nanowire system which consistsof a wet-chemically grown silver wire core surrounded by a SiO₂ shell ofcontrolled thickness, followed by an outer shell of highly luminescentCdSe nanocrystals [Yuri Fedulil,† Vasily Temnov,† Ulrike Woggon,† ElenaUstinovich,‡ and Mikhall Artemyev*‡, Exciton-Plasmon interaction in aComposite Metal-Insulator-Semiconductor Nanowire System, J. Am. Chem.Soc., 129 (48), 14939-14945, 2007]. For a SiO₂ spacer thickness of ˜15nm, they observed an efficient excitation of surface plasmons byexcitonic emission of CdSe nanocrystals. For small d, well below 10 nm,the emission is strongly suppressed (PL quenching), in agreement withthe expected dominance of the dipole-dipole interaction with the dampedmirror dipole [G. W. Ford and W. H. Weber, Electromagnetic interactionsof molecules with metal surfaces,” Phys. Rep. 113, 195-287 (1984)]. Fornanowire lengths up to ˜10 μm, the compositemetal-insulator-semiconductor nanowires ((Ag)SiO₂)CdSe act as awaveguide for ID-surface plasmons at optical frequencies with efficientphoton out coupling at the nanowire tips, which is promising forefficient exciton-plasmon-photon conversion and surface plasmon guidingon a submicron scale in the visible spectral range.

Experiments on colloidal solutions of Ag nanoparticles covered withJ-aggregates demonstrated the possibility of using the strong scatteringcross section and the enhanced field associated with surface plasmon togenerate stimulated emission from J-aggregate excitons with very lowexcitation powers. [Gregory A. Wurt,* Paul R. Evans, William Hendren,Ronald Atkinson, Wayne Dickson, Robert J. Pollard, and Anatoly V.Zayats, Molecular Plasmonics with Tunable Exciton-Plasmon CouplingStrength in J-Aggregate Hybridized Au Nanorod Assemblies, Nano Lett.,Vol. 7, No. 5, 1297, 2007]. Their coupling to surface plasmonsexcitations therefore provides a particularly attractive approach forcreating low-powered optical devices. This process can lead to efficientX-ray coupling for phototherapy. In addition, the coupling ofJ-aggregates with plasmonics structures presents genuine fundamentalinterest in the creation of mixed plasmon-exciton states.

Design, Fabrication and Operation of EPEP Probes

FIG. 18 shows various embodiments of EPEP probes of the presentinvention showing the exciton-plasmon coupling:

(A) a probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanoparticlecovered with a nanoshell of silica (or other dielectric material). Thesilica layer (or nanoshell) is designed to prevent quenching of theluminescence light emitted by the energy modulation agent particleexcited by X-ray. The metal nanoparticle (Au, Ag, etc) is designed toinduce plasmons that enhance the X ray excitation that subsequentlyleads to an increase in the energy modulation agent light emission,ultimately enhancing the efficiency of photoactivation, i.e.phototherapy. The structure of the nanoparticle can also be designedsuch that the plasmonics effect also enhances the energy modulationagent emission light. These processes are due to strong coupling betweenexcitons (in the energy modulation agent materials and plasmons in themetal nanoparticles; and

(B) a probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanoparticle viaa spacer (linker). The spacer is designed to prevent quenching of theluminescence light emitted by the energy modulation agent particleexcited by X-ray.

FIG. 19 shows yet further embodiments of EPEP probes of the presentinvention:

(A) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is covered with a nanoshell of silica (or otherdielectric material), which is covered by a layer of separatenanostructures (nano islands, nanorods, nanocubes, etc. . . . ) of metal(Au, Ag). The silica layer (or other dielectric material) is designed toprevent quenching of the luminescence light emitted by the EEC (alsoreferred to as energy modulation agent) particle excited by X-ray. Themetal nanostructures (Au, Ag, etc) are designed to induce plasmons thatenhance the X ray excitation that subsequently leads to an increase inthe EEC light emission, ultimately enhancing the efficiency ofphotoactivation, i.e. phototherapy. The structure of the nanoparticlecan also be designed such that the plasmonics effect also enhance theenergy modulation agent emission light. These processes are due tostrong coupling between excitons (in the energy modulation agentmaterials and plasmons in the metal nanostructures).

(B) a probe comprising a group of PA molecules in a particle bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The PA-containingparticle is covered with a layer of metallic nanostructures (Au, Ag).The metal nanostructures (Au, Ag, etc) are designed to induce plasmonsthat enhance the energy modulation agent light emission, ultimatelyenhancing the efficiency of photoactivation, i.e. phototherapy.

(C) a probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is covered with a nanoshell of silica (or otherdielectric material), which is covered by a layer of metallicnanostructures (Au, Ag). The silica layer (or other dielectric material)is designed to prevent quenching of the luminescence light emitted bythe energy modulation agent particle excited by X-ray. The metalnanostructures (Au, Ag, etc) are designed to induce plasmons thatenhance the X ray excitation that subsequently leads to an increase inthe energy modulation agent light emission, ultimately enhancing theefficiency of photoactivation, i.e. phototherapy. In addition, thePA-containing particle is covered with a layer of metallicnanostructures (Au, Ag). The metal nanostructures (Au, Ag, etc) aredesigned to induce plasmons that enhance the EEC light emission,ultimately enhancing the efficiency of photoactivation, i.e.phototherapy.

Hybrid EPEP Nano-Superstructures

EPEP probes can also comprise hybrid self-assembled superstructures madeof biological and abiotic nanoscale components, which can offerversatile molecular constructs with a spectrum of unique electronic,surface properties and photospectral properties for use in phototherapy.

Biopolymers and nanoparticles can be integrated in superstructures,which offer unique functionalities because the physical properties ofinorganic nanomaterials and the chemical flexibility/specificity ofpolymers can be used. Noteworthy are complex systems combining two typesof excitations common in nanomaterials, such as excitons and plasmonsleading to coupled excitations. Molecular constructs comprising buildingblocks including metal, semiconductor nanoparticles (NPs), nanorods(NRs) or nanowires (NWs) can produce EPEP probes with an assortment ofphotonic properties and enhancement interactions that are fundamentallyimportant for the field of phototherapy. Some examples of assemblies ofsome NW nanostructures and NPs have been reported in biosensing.Nanoscale superstructures made from CdTe nanowires (NWs) and metalnanoparticles (NPs) are prepared via bioconjugation reactions.Prototypical biomolecules, such as D-biotin and streptavidin pair, wereutilized to connect NPs and NWs in solution. It was found that Au NPsform a dense shell around a CdTe NW. The superstructure demonstratedunusual optical effects related to the long-distance interaction of thesemiconductor and noble metal nanocolloids. The NW□NP complex showed5-fold enhancement of luminescence intensity and a blue shift of theemission peak as compared to unconjugated NW. [Jaebeom Lee,† AlexanderO. Govorov,‡ John Dulka,‡ and Nicholas A. Kotov*,†, Bioconjugates ofCdTe Nanowires and Au Nanoparticles: Plasmon-Exciton Interactions,Luminescence Enhancement, and Collective Effects, Nano Lett., Vol. 4,No. 12, 2323, 2004].

To the present inventors' knowledge, these advanced concepts have notbeen applied to phototherapy and EPEP probes comprising superstructuresfrom NPs, NRs and NWs are still a new unexplored territory ofphototherapy.

FIG. 20 shows various embodiments of EPEP probes of the presentinvention comprising superstructures of NPs, NWs and NRs.:

(A) a probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanowire (ornanorod) covered with a nanoshell cylinder of silica (or otherdielectric material). The silica nanoshells cylinder is designed toprevent quenching of the luminescence light emitted by the energymodulation agent particle excited by X-ray. The metal nanoparticle (Au,Ag, etc) is designed to induce plasmons that enhance the X rayexcitation that subsequently leads to an increase in the energymodulation agent light emission, ultimately enhancing the efficiency ofphotoactivation, i.e. phototherapy. The structure of the nanoparticlecan also be designed such that the plasmonics effect and/or theexciton-plasmon coupling (EPC) effect also enhances the energymodulation agent emission light. These processes are due to strongcoupling between excitons (in the energy modulation agent materials andplasmons in the metal nanoparticles; and

(B) a probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanoparticlesvia a spacer (linker). The spacer is designed to prevent quenching ofthe luminescence light emitted by the energy modulation agent particleexcited by X-ray. Same effect as above in (A)

FIG. 21 shows another set of embodiments of EPEP probes of the presentinvention comprising superstructures of NPs, NWs and NRs andbioreceptors (antibodies, DNA, surface cell receptors, etc.). The use ofbioreceptors to target tumor cells has been discussed previously abovein relation to PEPST probes. Note that in this embodiment the PAmolecules are attached along the NW axis in order to be excited by theemitting light form the NWs.

FIG. 22 shows another embodiment of EPEP probes of the present inventioncomprising superstructures of NPs linked to multiple NWs.

For some embodiments, by adding metal nanostructures designed tointeract specifically with the excitons in the energy modulation agentsystem, there are significant improvements:

(1) an additional radiative pathway from exciton to photon conversion isintroduced

(2) the metal nanostructures can be designed to amplify (due to theplasmonics effect) the excitation radiation (e.g., X-ray) and/or theemission radiation (e.g., UV or visible) to excite the photo-active (PA)molecule, thereby enhancing the PA effectiveness.

Various metallic nanostructures that can be used in EPEP probeembodiments of the present invention are the same as those illustratedin FIG. 9 for the PEPST probes.

EPEP Probes with Microresonators

In a preferred embodiment the energy modulation agent system can bedesigned to serve also as a microresonator having micron or submicronsize. Lipson et al described a resonant microcavity and, moreparticularly, to a resonant microcavity which produces a stronglight-matter interaction [M. Lipson: L. C. Kimerling; Lionel C, Resonantmicrocavities, U.S. Pat. No. 6,627,923, 2000]. A resonant microcavity,typically, is formed in a substrate, such as silicon, and has dimensionsthat are on the order of microns or fractions of microns. The resonantmicrocavity contains optically-active matter (i.e., luminescentmaterial) and reflectors which confine light in the optically-activematter. The confined light interacts with the optically-active matter toproduce a light-matter interaction. The light-matter interaction in amicrocavity can be characterized as strong or weak. Weak interactions donot alter energy levels in the matter, whereas strong interactions alterenergy levels in the matter. In strong light-matter interactionarrangements, the confined light can be made to resonate with theseenergy level transitions to change properties of the microcavity.

Experimental Methods

Preparation of Nanoparticles (Ag, Au)

There many methods to prepare metal nanoparticles for EPEP or PEPSTprobes. Procedures for preparing gold and silver colloids includeelectroexplosion, electrodeposition, gas phase condensation,electrochemical methods, and solution-phase chemical methods. Althoughthe methodologies for preparing homogeneous-sized spherical colloidalgold populations 2-40 nm in diameter are well known [N. R. Jana, L.Gearheart and C. J. Murphy, Seeding growth for size control of 5-40 nmdiameter gold nanoparticles. Langmuir 17 (2001), pp. 6782-6786], andparticles of this size are commercially available. An effective chemicalreduction method for preparing populations of silver particles (withhomogeneous optical scattering properties) or gold particles (withimproved control of size and shape monodispersity) is based on the useof small-diameter uniform-sized gold particles as nucleation centers forthe further growth of silver or gold layers.

A widely used approach involves citrate reduction of a gold salt toproduce 12-20 nm size gold particles with a relatively narrow sizedistribution. The commonly used method for producing smaller goldparticles was developed by Brust et al [Brust, M.; Walker, M.; Bethell,D.; Schifin, D. J.; Whyman, R. Chem. Commun. 1994, 801]. This method isbased on borohydride reduction of gold salt in the presence of analkanethiol capping agent to produce 1-3 nm particles. Nanoparticlesizes can be controlled between 2 and 5 nm by varying the thiolconcentration, [Hostetler, M. J.; Wingate, J. E; Zhong, C. J.; Harris,J. E.; Vachet, R. W.; Clark; M. R.; Londono, J. D.; Green, S. J.;Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N.D.; Murray, R. W. Langmuir 1998, 14, 17]. Phosphine-stabilized goldclusters have also been produced and subsequently converted tothiol-capped clusters by ligand exchange in order to improve theirstability [Schmid, G.; Pfeil, R; Boese, R; Bandrmann, F.; Meyer, S.;Calis, G. H. M; van der Velden, J. W. A. Chem. Ber. 1981, 114, 3634;Warner, M. G.; Reed, S. M.; Hutchison, J. E. Chem. Mater. 2000, 12,3316.] and phosphine-stabilized monodispersed gold particles wereprepared using a similar protocol to the Brust method [Weare, W. W.;Reed S. M.; Warner, M. G.; Hutchison, J. E. J. Am. Chem. Soc. 2000, 122,12890]. See also recent review: Ziyi Zhong, Benoit¹ Male, Keith B.¹Luong, John H. T., More Recent Progress in the Preparation of AuNanostrutures, Properties and Applications Analytical Letters; 2003,Vol. 36 Issue 15, p 3097-3118]

Fabrication of Nanoparticle of Metal Coated with Nanoshells of Dyes

The fabrication of metal nanoparticles coated with nanoshells of dyemolecules can be performed using the method described by Masuhara et al[AKITO MASUHARA, SATOSHI OHHASHIy, HITOSHI KASAI; SHUJI OKADA,FABRICATION AND OPTICAL PROPERTIES OF NANOCOMPLEXES COMPOSED OF METALNANOPARTICLES AND ORGANIC DYES, Journal of Nonlinear Optical Physics &Materials Vol. 13, No. 3 & 4 (2004) 587-592]. Nanocomplexes composed ofAg or Au as a core and3-carboxlymethyl-5-[2-(3-octadecyl-2-benzoselenazolinylidene)ethylidene]rhodanine(MCSe) or copper (II) phthalocyanine (CuPc) as a shell are prepared bythe co-reprecipitation method. In the case of Ag-MCSe nanocomplexes, 0.5mM acetone solution of MCSe are injected into 10 ml of Ag nanoparticlewater dispersion, prepared by the reduction of AgNO₃ using NaBH₄:Au-MCSe nanocomplexes are also fabricated in a similar manner. A waterdispersion of Au nanoparticles was prepared by the reduction of HAuCl₄using sodium citrate. Subsequently, 2 M NH₄OH (50 μl) was added and themixture was thermally treated at 50° C. This amine treatment oftenstimulates the J-aggregate formation of MCSe.6 Ag-CuPc and Au-CuPcnanocomplexes were also fabricated in the same manner: 1 mM1-methyl-2-pyrrolidinone (NMP) solution of CuPc (200 μl) was injectedinto a water dispersion (10 ml) of Ag or Au nanoparticles.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Gold Nanoshell Preparations with Dielectric Cores:

Materials:

Yttrium oxide nanoparticles (e.g., 99.9% purity, 32-36 nm averagediameter, cubic crystal structure) were obtained from Nanostructured andAmorphous Materials, Inc. (Houston, Tex.). Tri-arginine(H-Arg-Arg-Arg-OH) acetate was obtained from Bachem (Torrance, Calif.),and gold tribromide (AuBr₃) was obtained from Alfa Aesar (Ward Hill,Mass.). Dimethyl sulfoxide (DMSO) was purchased from CalBioChem (LaJolla, Calif.) and was used as received. A cysteine-modified version ofthe TAT peptide (residues 49-57, sequenceArg-Lys-Lys-Arg-Arg-Arg-Gln-Arg-Arg-Cys-CONH₂ (SEQ ID NO: 1), molecularweight 1442 g/mol, hereafter referred to as “TAT”) was obtained fromSynBioSci (Livermore, Calif.).Succinimidyl-[4-(psoralen-8-yloxy)]butyrate (SPB) was obtained fromPierce (Rockford, Ill.), and Marina Blue, Alexa 350 and Alexa 546 NHSesters were obtained from Invitrogen (Carlsbad, Calif.). Ultrapure 18.2MΩ deionized (DI) water purified with a Millipore Synergy filtrationsystem (Millipore, Billerica, Mass.) was used to make all solutions.

Yttrium Oxide Dispersion:

Tip sonication was used to disperse autoclaved Y₂O₃ nanoparticles at 10mg/mL in 10 mM tri-arginine solution which had been pre-filtered at 0.22microns. Following moderate mixing in a sealed, sterile container on astir plate for 24 hours to allow tri-arginine attachment and improvedY₂O₃ dispersion, the solution was centrifuged at 8200 relativecentrifugal force (RCF) to remove fused particles and large aggregates.

Gold Shell Formation:

Supernatant from the initial Y₂O₃ dispersion was diluted 1:1 (v/v) with5.7 mM AuBr₃ dissolved in sterile DI water and pre-filtered at 0.22microns, then exposed to high-intensity fluorescent light (CommercialElectric, Model 926) for 16 hours in a sealed, sterile glass containerwith moderate mixing. During the time course of this photochemicalprocess, the reddish-brown AuBr₃ solution turned yellow immediatelyafter addition of the Y₂O₃ in tri-arginine; became clear and visuallycolorless; then developed an intense purple color as Au shells formed onthe Y₂O₃ cores. In the absence of the Y₂O₃ cores, neither the intensepurple color associated with plasmonic absorption by gold nanoshells northe deep red color associated with solid gold nanoparticles appears. Useof heat rather than light in the presence of Y₂O₃ particles tends toproduce a large number of solid gold nanoparticles rather than or inaddition to core-shell structures, as evidenced by strong absorption at˜530 nm.

Particle Functionalization with TAT:

Gold-coated Y₂O₃ nanoparticles were centrifuged at 16k RCF for 15minutes, and the pellet was re-dispersed in a 50% volume of sterile DIwater by a short tip sonication. The particles were further purified bytwo additional centrifugations at 16k RCF for 15 minutes each, withredispersion in a 100% volume of sterile DI water following the secondcentrifugation and final redispersion in a 100% volume of 1 mg/mL (0.7mM) TAT peptide dissolved in sterile DI water and pre-filtered at 0.22microns.

This solution was vigorously mixed at room temperature for one hour toallow thiol anchoring to the gold shell via the c-terminal cysteineresidue. Variations in the TAT concentration, temperature and reactiontime can all affect the extent of surface coverage and the potential forfurther functionalization.

Peptide Functionalization with Dye Molecules:

The TAT-functionalized, gold-coated Y₂O₃ particles were purified bytriplicate centrifugation at 16k RCF, with the first two re-dispersionsin sterile DI water and the final re-dispersion in sterile 100 mMbicarbonate buffer at pH 9.0. Each NHS ester (SPB, Alexa 350, MarinaBlue and Alexa 546) was dissolved at 10 mg/mL in dimethyl sulfoxide(DMSO), and 100 microliters of a given NHS-functionalized dye were addedto a 1 mL aliquot of TAT-functionalized, gold-coated Y₂O₃. The solutionswere reacted for one hour at room temperature in the dark with vigorousmixing to allow attachment of dye molecules to primary amines along theTAT peptide (such as the attachment of N terminus and the lysine sidechains).

The psoralen-functionalized nanoparticles were centrifugally cleanedusing a 1:1 volume of DMSO in water to remove any residual SPB crystals,then all dye-functionalized core-shell nanoparticles were purified bytriplicate centrifugation at 16k RCF for 15 minutes. Each centrifugationstep was followed by re-dispersion in a 100% volume of sterile DI water.Presuming removal of 95+% of non-attached dye molecules during eachcentrifugation step, no more than 0.01% of the unbound dye is estimatedto remain in the final solution.

Nanoparticle Characterization:

Transmission electron microscopy (TEM) provides additional evidence forthe presence of gold-coated Y₂O₃ particles. FIG. 6E, for example, showsa representative TEM image of as purchased Y₂O₃ nanoparticles. Theparticles are quite polydisperse, but exhibit an average diameter ofapproximately 35 nm. FIG. 10F shows similar images for Y₂O₃ particlescoated with a gold shell using the synthetic procedure described above.Like the underlying Y₂O₃ cores, the gold-coated yttrium oxide particlesare somewhat polydisperse with an average diameter of approximately 50nm.

Perhaps the most conclusive demonstration that these nanoparticles arein fact gold-coated Y₂O₃ comes from comparison of X-ray diffraction data(XRD). FIG. 6G shows diffractograms for both the initial cubic Y₂O₃nanoparticles (lower trace) and the final gold-coated core-shellparticles (upper trace). Strong peaks at 2θ=29, 33.7, 48.5 and 57.5degrees in the lower trace are indicative of cubic Y₂O₃. The mostpronounced features in the upper trace are two gold-associated peaks at2θ=38.2 and 44.4 degrees. In addition, the four strongest cubic Y₂O₃peaks at 2θ=29, 33.7, 48.5 and 57.5 degrees are also visiblysuperimposed on the baseline diffractogram from the gold nanoshells. Thereason for the broadening of the Y₂O₃ peak at 2θ=29 degrees is notdefinite, but may be a result of gold-Y₂O₃ interactions or,alternatively, the preferential size-selection of small Y₂O₃ particlesduring the 8200 RCF centrifugation used to remove large Y₂O₃ particlesand aggregates.

Gold Colloidal Nanopartides:

a. Synthesis of Gold Nanoparticles

The Frens method (see G. Frens, Nat. Phys. Sci. 241 (1973) 20, theentire contents of which are incorporated herein by reference) can beused to synthesize gold nanoparticles. In this process, 5.0×10⁻⁶ mol ofHAuCl₄ was dissolved in 19 mL of deionized water. The resulting solutionwas faintly yellow. The solution was heated and vigorously stirred in arotary evaporator for 45 minutes. One mL of 0.5% sodium citrate wasadded, and the solution was stirred for an additional 30 minutes.Addition of sodium citrate has multiple purposes. First, citrate acts asa reducing agent. Second, citrate ions that adsorb onto the goldnanoparticles introduce surface charge that stabilizes the particlesthrough charge repulsion, thus preventing nanocluster formation.

b. Synthesis of Gold Nanoparticles Having 15-nm Diameter

Two mL of 1% gold chloride in 90 mL DI water was heated to 80° C. for 15minutes, then 80 mg sodium citrate in 10 ml DI water was added. Thesolution was boiled and vigorously stirred for 30 minutes. FIG. 10Hshows pictures of ˜15-nm gold nanoparticles prepared using citratereduction.

c. Synthesis of 30-nm Gold Nanoparticles

Two mL of 1% HAuCl₄ solution in a 100-mL round-bottom flask were mixedwith 20 mg of sodium citrate, then boiled and vigorously stirred for 30minutes. FIG. 10I shows TEM images of 30-nm gold nanoparticles preparedusing the citrate reduction technique.

d. Synthesis of 60-nm Gold Nanoparticles

Two mL of 1% HAuCl4 in 100 mL of water were mixed with 10 mg of sodiumcitrate. The solution was boiled and vigorously stirred for 30 minutes.FIG. 10J shows TEM pictures of 60-nm gold nanoparticles prepared usingthe citrate reduction technique.

e. Use of Hydrazine Monohydrate as a Reducing Agent:

100 microliters (0.1 mL) of 12 millimolar gold chloride solution wasdiluted with 80 ml H₂O in a beaker. The initial pH of the gold solutionwas 3.67. The temperature of the solution was increased to 80° C. for 30minutes, at which point 0.3 mL hydrazine monohydrate was added to thegold solution. The solution pH shifted to 7.64. Over time, gold solutionchanged to a very light pink color. FIG. 10K shows TEM pictures of 30-nmgold nanoparticles prepared using the hydrazine monohydrate reductiontechnique.

Colloidal Silver Nanoparticles:

Use of Sodium Citrate as a Reducing Agent:

In this method, 50 mL of a 10⁻³ M AgNO₃ aqueous solution was heated toboiling. Then, 1 mL of a 1% trisodium citrate (C₆H₅O₇Na₃) was added tothe solution, and the solution was maintained at boiling for 1 hourbefore being allowed to cool. The resultant colloidal mixture exhibiteda dark grey color.

Use of Hydroxylamine Hydrochloride as a Reducing Agent:

A colloidal solution was formed by dissolving 0.017 g of silver nitrate(AgNO₃) in 90 mL water. 21 mg of hydroxylamine hydrochloride (NH₂OH.HCl)was dissolved in 5 mL water and 4.5 ml of 0.1 M sodium hydroxide wasadded. This mixture was added to the AgNO₃ solution. Very rapidly, (e.g,just in a few seconds), a grey-brown solution appeared.

Use of Sodium Borohydride as a Reducing Agent:

Aqueous solutions containing 10 mL 10⁻³ M AgNO₃ and 30 mL 10⁻³ M NaBH₄were mixed under ice-cooled conditions. The AgNO₃ solution was addeddropwise to the NaBH₄ solution with vigorous stirring. The resultantmixture was allowed to age 1 hour before stirring the resultant mixtureagain for 10 minutes.

Metallic/Dielectric, Multi-Layer, Core-Shell Nanoparticles:

Au Nanoparticles Coated with Ag or an Nanoparticles Coated with Au:

Core-shell nanoparticles such as gold-coated silver nanoparticles andsilver-coated gold nanoparticles have been synthesized in an aqueousmedium using CTAB as a surfactant and ascorbic acid as a reducing agent.Core nanoparticles (i.e. Au or Ag) were prepared using the aboveprocedures, then coated with secondary, tertiary, etc. shells.

For example, spherical gold nanoparticles (˜15 nm) were prepared byboiling HAuCl₄ in the presence of sodium citrate. For coating gold witha layer of silver, 1 mL of 0.1 M ascorbic acid solution, 0.5 mL of 10 mMAgNO₃ solution, and 0.5 mL of the previously formed Au colloid weresequentially added to 20 mL of a 50 mM CTAB solution. Subsequently, 0.1mL of 1.0 M NaOH was added dropwise, which led to a fast color change(from red to yellow). FIG. 6M shows TEM images of Au nanoparticlescoated with Ag.

A similar procedure was used to prepare Ag nanoparticles coated with Au.

Au@Ag@Au@Ag Multi Shell Nanoparticles:

Multishell nanoparticles such as Au@Ag@Au@Ag were prepared using CTAB asa surfactant, and ascorbic acid and NaOH as reducing agents. Sphericalgold nanoparticles (˜15 nm) were prepared by boiling HAuCl₄ in thepresence of sodium citrate. To coat gold cores with a layer of silver,20 mL of a 50 mM CTAB, 1 mL of 0.1 M ascorbic acid, 0.5 mL of 10 mMAgNO₃, and 0.5 mL of the Au colloid were sequentially mixed.Subsequently, 0.1 mL of 1.0 M NaOH was added in a dropwise manner, whichled to a fast color change from red to yellow.

Then, another gold layer was coated by mixing 20 mL of the Ag-coated Aucolloid in water with 1 mL of the ascorbic acid solution. The resultingmixture was then added to 0.05 mL of 0.10 M HAuCl₄ in a dropwise manner.The solution color changed to deep blue at this stage. Subsequently, anouter silver shell was formed on the previously formed Au@Ag@Aunanoparticles by mixing 20 mL of colloid with 0.5 mL 10 mM AgNO₃followed by drop wise addition of 0.2 mL of 1.0 M NaOH. The solutionthen showed a color change to orange. FIG. 10N shows TEM images ofAu@Ag@Au@Ag multi-shell nanoparticles.

All of the above core-shell nanoparticle solutions were stable insolution.

Chemical Synthesis of Multi-Layer Core-Shell Structures Using Y₂O₃

To deposit multiple shells on Y₂O₃ nanoparticles, Y₂O₃ nanoparticleswere initially coated with Ag via UV photoreduction in a proceduresimilar to that discussed above for gold shells. In the presentinvention, a number of approaches can be utilized for the addition of agold shell. These include 1) a sodium citrate process, 2) a sodiumborohydride reduction, 3) a hydrazine monohydrate reduction, 4) asolution containing hydroxyl amine and NaOH, and 5) a mixture of CTAB,ascorbic acid, and NaOH.

Use of Sodium citrate as a Reducing Agent:

A typical experiment used 0.1 to 1 mL of Y₂O₃ coated with Ag (˜50 nm), 1to 3 mL of 2.5 H10⁻³ M HAuCl₄, and 50 mL distilled water in a 100 mlround bottom flask. This solution was boiled with constant stirring, and3 mL of 1 wt % sodium citrate was added. The resultant colloidalsolution color became black with a pH of approximately pH 6.5. Thesolution was stirred for another 15 min and then allowed to stand.

Use of Sodium Borohydride as Reducing Agent:

A typical experiment used 0.1 to 1 mL of Y₂O₃ coated with Ag (˜50 nm), 1to 3 mL of 2.5 H10⁻³ M HAuCl₄, and 50 mL distilled water in a 100 mLround bottom flask. Under constant stirring this solution was boiledprior to addition of 0.1 to 1 mL of 0.1 M NaBH₄ solution. The resultantcolloidal solution became black and aggregated within a few minutes.

Probe for Measurement of Apoptosis with the PDT Drug ALA

A method has been developed using nanosensors that can be used toevaluate the effectiveness of PEPST probes. Although one can useconventional methods (not requiring nanosensors), we describe thenanosensor method previously developed [P. M. Kasili, J. M. Song, and T.Vo-Dinh, “Optical Sensor for the Detection of Caspase-9 Activity in aSingle Cell”, J. Am. Chem. Soc., 126, 2799-2806 (2004)]. The methodcomprises measuring caspases activated by apoptosis induced by thephotoactive drugs. In this experiment, we measure two sets of cells Iand II. Set I is treated with the drug ALA and set II is treated by thedrug ALA conjugated to a PEPST probe described in the previous section.By comparing the results (amount of Caspases detected), one can evaluatethe efficiency of the PEPST-ALA drug compared to ALA alone.

In the classical model of apoptosis, caspases are divided into initiatorcaspases and effector caspases according to their function and theirsequence of activation. Initiator caspases include caspase-8, -9, whileeffector caspases include, caspases-3, -6 and -7. The activation ofcaspases is one of the earliest biomarkers of apoptosis making caspasesan early and ideal target for measuring apoptosis. Apoptosis, orprogrammed cell death, is a mode of cell death characterized by specificmorphological and biochemical features. The results obtained in theseexperiments can be used to evaluate the effectiveness ofphototherapeutic drugs that induce apoptosis (e.g. PDT drugs). Sincecaspases play a central role in the induction of apoptosis,tetrapeptide-based optical nanosensors were used to determine their rolein response to a photodynamic therapy (PDT) agent, 6-aminolevulinic acid(ALA) in the well-characterized human breast carcinoma cell line, MCF-7.MCF-7 cells were exposed to the photosensitizer ALA to explore ALA-PDTinduced apoptosis by monitoring caspase-9 and caspase-7 activity.Caspase-9 and caspase-7 protease activity was assessed in single livingMCF-7 cells with the known caspase-9 and caspase-7 substrates,Leucine-aspartic-histidine-glutamic acid 7-amino-4-methylcoumarin(LEHD-AMC) and aspartic-glutamic acid-valine-aspartic acid7-amino-4-methylcoumarin (DEVD-AMC) respectively, covalently immobilizedto the nanotips of optical nanosensors. Upon the induction of apoptosis,activated target caspases recognize the tetrapeptide sequence andspecifically cleaves it. The recognition of substrate by caspases isimmediately followed by a cleavage reaction yielding the fluorescent AMCwhich can be excited with a Helium-Cadmium (HeCd) laser to generate ameasurable fluorescence signal. By comparing the fluorescence signalgenerated from AMC within cells with activated caspases and from thosewith inactive caspases, we are able to successfully detect caspaseactivity within a single living MCF-7 cell.

Chemicals and Reagents

δ-aminolevulinic acid (ALA), phosphate buffered saline (PBS),hydrochloric acid (HCl), nitric acid (HNO₃),Glycidoxypropyltrimethoxysilane (GOPS), 1,1′-Carbonyldiimidazole (CDI),and anhydrous acetonitrile were purchased from Sigma-Aldrich, St. Louis,Mo. Caspase-9 substrate, LEHD-7-amino-4-methylcoumarin (AMC), Caspase-7substrate, DEVD-7-amino-4-methylcoumarin (AMC), 2× reaction buffer,dithiothreitol (DTT), and dimethylsulfoxide (DMSO) were purchased fromBD Biosciences, Palo Alto. Calif.

Cell Lines

Human breast cancer cell line, MCF-7, was obtained from American TypeCulture Collection (Rockville, Md., USA, Cat-no. HTB22). MCF-7 cellswere grown in Dulbecco's Modified Eagle's Medium ((DMEM) (Mediatech,Inc., Herndon, Va.)) supplemented with 1 mM L-glutamine (Gibco, GrandIsland, N.Y.) and 10% fetal bovine serum (Gibco, Grand Island, N.Y.).Cell culture was established in growth medium (described above) instandard T25 tissue culture flasks (Corning, Corning, N.Y.). The flaskswere incubated in a humidified incubator at 37° C., 5% CO2 and 86%humidity. Cell growth was monitored daily by microscopic observationuntil a 60-70% state of confluence was achieved. The growth conditionswere chosen so that the cells would be in log phase growth duringphotosensitizer treatment with ALA, but would not be so close toconfluence that a confluent monolayer would form by the termination ofthe chemical exposure. In preparation for experiments, cells wereharvested from the T25 flasks and 0.1 ml (10⁵ cells/ml) aliquots wereseeded into 60 mm tissue culture dishes (Corning Costar Corp., Corning,N.Y.) for overnight attachment. The MCF-7 cells were studied as fourseparate groups with the first group, Group I, being the experimental,exposed to 0.5 mM ALA for 3 h followed by photoactivation([+]ALA[+]PDT). This involved incubating the cells at 37° C. in 5% CO₂for 3 h with 0.5 mM ALA. Following incubation the MCF-7 cells wereexposed to red light from a HeNe laser (λ 632.8 nm, <15 mW, MellesGriot, Carlsbad, Calif.) positioned about 5.0 cm above the cells fbrfive minutes at a fluence of 5.0 mJ/cm² to photoactivate ALA andsubsequently induce apoptosis. The second and third groups, Group II andII respectively, served as the “treated control” and were exposed to 0.5mM ALA for 3 hours without photoactivation ([+]ALA[−]PDT) andphotoactivation without 0.5 mM ALA ([−]ALA[+]PDT]) respectively. Thefourth group, Group IV was the “untreated control,” which receivedneither ALA nor photoactivation ([−]ALA[−]PDT

Preparation of Enzyme Substrate-Based Optical Nanosensors

Briefly, this process involved cutting and polishing plastic clad silica(PCS) fibers with a 600-μm-size core (Fiberguide Industries, Stirling,N.J.). The fibers were pulled to a final tip diameter of 50 nm and thencoated with ˜100 nm of silver metal (99.999% pure) using a thermalevaporation deposition system (Cooke Vacuum Products, South Norwalk,Conn.) achieving a final diameter of 150 nm. The fused silica nanotipswere acid-cleaned (HNO₃) followed by several rinses with distilledwater. Finally, the optical nanofibers were allowed to air dry at roomtemperature in a dust free environment. The nanotips were then silanizedand treated with an organic coupling agent, 10%Glycidoxypropyltrimethoxysilane (GOPS) in distilled water. Thesilanization agent covalently binds to the silica surface of thenanotips modifying the hydroxyl group to a terminus that is compatiblewith the organic cross-linking reagent, 1′1, Carbonyldiimidazole (CDI).The use of CDI for activation introducing an imidazole-terminal groupwas particularly attractive since the protein to be immobilized could beused without chemical modification. Proteins bound using this procedureremained securely immobilized during washing or subsequent manipulationsin immunoassay procedures, as opposed to procedures that use adsorptionto attach proteins. The silanized and activated nanotips for measuringcaspase-9 activity were immersed in a solution containing DMSO, 2×reaction buffer, PBS, and LEHD-AMC, and allowed to incubate for 3 h at37° C., while those for measuring caspase-7 activity were immersed in asolution containing DMSO, 2× reaction buffer, PBS, and DEVD-AMC, andallowed to incubate for 3 h at 37° C.

Measurement System and Procedure

A schematic representation of the experimental setup used in this workis described in a previous work [P. M. Kasili, J. M. Song, and T.Vo-Dinh, “Optical Sensor for the Detection of Caspase-9 Activity in aSingle Cell”, J. Am. Chem. Soc., 126, 2799-2806 (2004)]. The componentsincluded a HeCd laser (Omnichrome, <5 mW laser power) for excitation, anoptical fiber for delivery of excitation light to the opticalnanosensor, a Nikon Diaphot 300 inverted fluorescence microscope (Nikon,Inc., Melville, N.Y.), a photon counting photomultiplier tube (PMT) anda PC for data acquisition and processing. This experimental set-up, usedto probe single cells, was adapted for this purpose from a standardmicromanipulation and microinjection apparatus. The Nikon Diaphot 300inverted microscope was equipped with a Diaphot 300/Diaphot 200Incubator to maintain the cell cultures at 37° C. on the microscopestage, during these experiments. The micromanipulation equipmentconsisted of MN-2 (Narishige Co. Ltd., Tokyo, Japan) Narishigethree-dimensional manipulators for coarse adjustment, and NarishigeMMW-23 three-dimensional hydraulic micromanipulators for fineadjustments. The optical nanosensor was mounted on a micropipette holder(World Precision Instruments, Inc., Sarasota, Fla.). The 325 nm laserline of a HeCd laser was focused onto a 600-μm-delivery fiber that isterminated with a subminiature A (SMA) connector. The enzymesubstrate-based optical nanosensor was coupled to the delivery fiberthrough the SMA connector and secured to the Nikon inverted microscopewith micromanipulators. To record the fluorescence generated by AMCmolecules at the nanotips, a Hamamatsu PMT detector assembly (HC125-2)was mounted in the front port of the Diaphot 300 microscope. Thefluorescence emitted by AMC from the measurement made using single livecells was collected by the microscope objective and passed through a330-380 nm filter set and then focused onto a PMT for detection. Theoutput from the PMT was recorded using a universal counter interfaced toa personal computer (PC) for data treatment and processing.

In Vitro Determination of Caspase Activity

After incubation using the following treatment groups, group (I)-[+]ALA[+]PDT, group II -[+]ALA[−]PDT, group III -[−]ALA[+]PDT, andgroup IV -[−]ALA[−]PDT, MCF-7 cells were washed with PBS solution, pH7.4, and then resuspended in lysis buffer (100 mM HEPES, pH 7.4, 10%sucrose, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS), 1 mMEDTA, 10 mM dithiothreitol (DTT), 1 mMphenylmethylsulphonyl fluoride (PMSF), 10 mg/ml pepstatin, 10 mg/mlleupeptin) and left on ice for 45 minutes. The cells were thenrepeatedly passed through a syringe with a 25-gauge needle until most ofthe cell membrane was disrupted, and centrifuged at 1500 RPM for 10 min.Activity of caspases was measured using the fluorogenic substratepeptides; LEHD-AMC for caspase-9 and DEVD-AMC for caspase-7. The releaseof AMC was measured after incubating optical nanosensors in picofugetubes containing the cell lysates from the various treatment groups andusing a HeCd laser (excitation 325 nm) to excite AMC. Caspase activitywas expressed as fluorescence intensity of AMC as a function ofequivalent nanomoles of LEHD-AMC and DEVD-AMC respectively.

The results of the in vitro measurement of caspase-9 and caspase-7activity were plotted. The curves for each fluorescent measurement ofAMC were plotted for each as a function of AMC concentration. Caspase-9activity was determined by incubation of optical nanosensors with thesubstrate LEHD-7-amino-4-methylcoumarin (AMC) in cell lysate (˜10⁵cells) obtained from the following treatment groups; group I, II, I andIV, described earlier in the article. The release of AMC was measuredafter excitation using HeCd laser (325 nm) and collecting thefluorescence signal using a 380 nm longpass filter. The peak emissionwavelength of AMC is about 440 nm. Likewise, Caspase-7 activity wasdetermined by incubation in cell lysate (˜10⁵ cells) obtained from thefollowing treatment groups I, II, I, and IV. The release of AMC wasmeasured after excitation using a HeCd laser (325 nm) and collecting thefluorescence signal using a 380 nm longpass filter.

In this experiment, we measure two sets of cells I and II: (1) Set I istreated with the drug ALA and (2) set II is treated by the drug ALAconjugated to a PEPST probe described in the previous section. Bycomparing the results (amount of caspase detected), one can evaluate theefficiency of the PEPST-ALA drug compared to ALA alone.

Tagging and Labeling Applications

Besides the medical applications presented above, the nanotechnology ofthe invention has applications in other areas such as security andtagging operations where a primary light source, for example a NIR beamis focused and directed onto a target object. Applications of thesematerials include: (i) detecting and removing of counterfeit currencyfrom circulation, (ii) detecting and removing of counterfeit adulteratedproducts (e.g., fake drugs), (iii) tracing the origin of products (e.g.,alcohol, tobacco, firearms) and commodities (e.g., oil/gas tag andtrace), (iv) tagging controlled substances (e.g. military explosives) orrestricted technology (e.g. nuclear and communications technologies),(v) marking single source, high value commodities (e.g., specialtyfibers), and (vi) brand protection, and (vii) verifying the authenticityof documents, financial instruments (e.g. bearer bonds), and variousforms of identification. With the NIR beam incident on nanoparticles ofyttrium oxide for example, the yttrium oxide nanoparticles will emit inthe visible wavelength range which can then be detected by a hand-heldreader, a CCD camera, or a person's eyes. For example, 100 to 1,000milliwatt power of NIR light at wavelength at 980 nanometers,upconverters of the types described in this application show brightgreen emission, blue emission, or red emission to the naked eye,emission so bright that one has to turn away viewing directly theemission.

Alternatively or complementarily, the nanotechnology of the inventionhas applications in security and tagging operations where the primarylight source is X-ray excitation and UV/VIS/NIR readout is used for theviewing.

In conventional bar coding operations, a scanner is used to essentiallyread a series of black and white lines with the density and spacingsbeing indicative of a particular coded item. In this invention, theseprinted bar codes could make use of the nanocore emitters describedabove which offer the possibility of a multicolor emission from eithersingular or multiple infrared laser sources. Thus, the amount ofinformation that can be encoded into a traditional bar code area may begreatly increased. For example, specific color categorization couldintroduce completely different encodings for what would normally be thesame series of black and white lines. Further, combinations of differingcolor lines would permit further encoding of information even on top ofthe existing bar code lines which could be read by existing black andwhite imagers, adding information that would be indicative of theclasses of product, class of distributers, class of manufacturers,classes of retailers, etc., in the product distribution chain. In thisway, bar codes applied at the manufacture or food packager could be usedfor example in food product tracking safety and monitoring.

In these tagging and labeling applications, the invention provides asystem for identification of an object. The system includes a readablemedium (e.g., a paper product, a plastic product, and a glass productwhich may be a part of a security tag or a bar code on any product), ananoparticle included in or on the surface of the readable medium. Thenanoparticle, upon exposure to a first wavelength λ₁ of radiation, isconfigured to emit a second wavelength λ₂ of radiation having a higherenergy than the first wavelength λ₁. The second wavelength λ₂ is in atleast one of infrared, visible, and ultraviolet light to permitidentification of the object by detecting the second wavelength λ₂.

A metallic shell can encapsulate at least a fraction of thenanoparticle. As explained above, a radial dimension of the metallicshell can be set to a value where a surface plasmon resonance in themetallic shell resonates at a frequency which provides spectral overlapwith either the first wavelength λ₁, or the second wavelength λ₂. Thenanoparticle can more generally include a plurality of nanoparticles.

As such, the nanoparticles can be divided into multiple groups orcategories of different light-emitting nanoparticles. A first group canfor example exhibit visible emission upon interaction with the firstwavelength λ₁, while a second group can exhibit infrared emission uponinteraction with the first wavelength λ₁. In this embodiment, the firstgroup can be a part of a visible tag on the object, and the second groupcan be a part of an invisible tag on the object. Alternatively, thefirst group can exhibit visible emission upon interaction with the firstwavelength λ₁, while the second group can exhibit ultraviolet emissionupon interaction with the first wavelength λ₁. In this embodiment also,the first group can be a part of a visible tag on the object, and thesecond group can be a part of an invisible tag on the object.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Quality Control and Environmental Sensor Applications

In one embodiment of the invention, a plasmonic effect is also availablevia Raman scattering, thereby providing a “signature” for the psoralencompound being used as a drug. Accordingly, in one embodiment of thisinvention, Raman scattering from these attached recipients 6 can be usedas an indicator of the presence or absence of plasmonic shells 6, theproximity of the psoralen to the plasmonic shell, etc. As such, theRaman enhancement effects can be used as a diagnostic to identify eitherfor 1) quality control measures, 2) assay measurements, or 3) productidentification to determine the type of psoralen in use or to be used.

Raman spectroscopy was originally developed to study vibrational modesof molecules, and has proven to be a valuable tool for characterizinglattice vibrations, phonon modes, of nanocrystals. Raman analysis hasbeen shown to be fairly effective in identifying the differences in thelocal chemical and crystalline structure about certain crystallinesystems. Presently, diode lasers and CCD cameras with the spectraldispersion elements can be used to instantaneously take Raman spectrafrom a wide variety of materials with digital counting techniquesavailable capture instantaneously an entire Raman spectrum and derivethe spectrum with sufficient signal to noise ratios which once requiredby high precision grating instruments and photon counting detectors.

Further, since the surface plasmon effect is a resonance of electrons inthe metallic shell 4 being confined between the dielectric inner coreand the environmental dielectric material, the plasmon resonance will beaffected by the dielectric properties of the medium itself. Thus, in oneembodiment of the invention, the novel dielectric core/shell structuresare used in conjunction with a Raman instrument as an environmentalsensor.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Sterilization and Cold Pasteurization of aids

Table 1 included below shows appropriate intensities for germicidaldestruction with UV light irradiation.

TABLE 1 Germicidal energies needed to destroy Approximate intensity(μW/cm²) required for 99% destruction of microorganisms: Bacteria  10400 Protozoa (single celled organism) 105 000 Paramecium (slipper shapedprotozoa) 200 000 Chlorella (unicellular fresh-water alga)  13 000Flagellate(protozoan or alga with flagella)  22 000 Sporozoan (parasiticprotozoans) 100 000 Virus  8 000

As shown in FIG. 25A, an exemplary system according to one embodiment ofthe invention may have an initiation energy source 1 directed at medium4. Activatable agents 2 and energy modulation agents 3 are dispersedthroughout the medium 4. The initiation energy source 1 may additionallybe connected via a network 8 to a computer system 5 capable of directingthe delivery of the initiation energy. In various embodiments, theenergy modulation agents 3 are encapsulated energy modulation agents 6,depicted in FIG. 25A as silica encased energy modulation agents. Asshown in FIG. 25A, initiation energy 7 in the form of radiation from theinitiation energy source 1 permeates throughout the medium 4.

As discussed below in more detail, the initiation energy source 1 can bean external energy source or an energy source located at least partiallyin the medium 4. As discussed below in more detail, activatable agents 2and/or the energy modulation agents 3 can include plasmonics agentswhich enhance either the applied energy or the energy emitted from theenergy modulation agents 3 so as to directly or indirectly produce achange in the medium.

In various embodiments, the initiation energy source 1 may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. In these embodiments, down conversion is used to generateinternal light inside the medium. One example of such linearaccelerators is the SmartBeam™ IMRT (intensity modulated radiationtherapy) system from Varian medical systems (Varian Medical Systems,Inc., Palo Alto, Calif.). In other embodiments, the initiation energysource 1 may be commercially available components of X-ray machines ornon-medical X-ray machines. X-ray machines that produce from 10 to 150keV X-rays are readily available in the marketplace. For instance, theGeneral Electric Definium series or the Siemens MULTIX series are buttwo examples of typical X-ray machines designed for the medicalindustry, while the Eagle Pack series from Smith Detection is an exampleof a non-medical X-ray machine. As such, the invention is capable ofperforming its desired function when used in conjunction with commercialX-ray equipment.

In other embodiments, the initiation energy source 1 can be a radiofrequency or microwave source or infrared source (as discussed above)emitting electromagnetic waves at a frequency which permeates the mediumand which triggers or produces or enhances secondary radiant energyemission within the medium by interaction with the energy modulationelements 6 therein. In other embodiments, the initiation energy source 1can be an ultraviolet, visible, near infrared (NIR) or infrared (IR)emitter emitting at a frequency which permeates the medium 4 and whichtriggers or produces secondary radiant energy emission within medium 4by interaction with the energy modulation elements 6 therein.

FIG. 25B is a schematic depicting another system according to anotherembodiment of the invention in which the initiation energy source 1 ofFIG. 25A is directed to energy modulation elements 6 placed in thevicinity of a fluid medium 4 (e.g., a liquid or other fluid-like medium)and held inside a container 9. The container 9 is made of a materialthat is “transparent” to the radiation 7. For example, plastic, quartz,glass, or aluminum containers would be sufficiently transparent toX-rays, while plastic or quartz or glass containers would be transparentto microwave or radio frequency radiation. The energy modulationelements 6 can be dispersed uniformly throughout the medium or may besegregated in distinct parts of the medium or further separatedphysically from the medium by encapsulation structures 10, as describedbelow. A supply 11 provides the medium 4 to the container 9.

Alternatively, as shown in FIG. 25C, the luminescent particles could bepresent in the medium in encapsulated structures 10. In one embodiment,the encapsulated structures 10 are aligned with an orientation in linewith the external initiation energy source 1. In this configuration,each of the encapsulated structures 10 has itself a “line-of-sight” tothe external initiation energy source 1 shown in FIG. 25C without beingoccluded by other of the encapsulated structures 10. In otherembodiments, the encapsulated structures 10 are not so aligned in thatdirection, but could aligned perpendicular to the direction shown inFIG. 25C, or could be randomly placed. Indeed, supply of fluid medium 4could itself be used to agitate the encapsulated structures 10 and mixthe fluid medium 4 inside container 9.

The system of FIG. 25C may also be used without energy modulationagents. In this embodiment, the initiation energy source 1 can be forexample at an energy suitable for driving physical, chemical, and/orbiological processes in the fluid medium 4. The plasmonics agentsincluded in the encapsulated structures 10 effectively amplify the lightfrom the initiation energy source 1 as it interacts with the medium 4.

FIG. 25D is a schematic depicting a system according to anotherembodiment of the invention in which the initiation energy source isdirected a container enclosing a medium having energy modulation agentssegregated within the medium in a fluidized bed 20 configurations. Thefluidized bed 20 includes the encapsulated structures 10 in aconfiguration where a fluid to be treated is passed between theencapsulated structures 10. The encapsulated structures 10 can includeboth energy modulation agents and plasmonics agents as described herein.

In the either configuration of FIGS. 25C and 25D, the medium to betreated would flow by the encapsulated structures 10, or flow along withencapsulated structures 6, and the separation distance between theencapsulated structures 6, 10 would be set a distance smaller than theUV penetration depth in the medium.

In further embodiments of the invention, robotic manipulation devicesmay also be included in the systems of FIGS. 25A, 25B, 25C, and 25D forthe purpose of delivering and dispersing the energy modulation elements6 in medium 4 or for the purpose of removing old product and introducingnew product for treatment into the system.

A suitable light source (such as one of the X-ray sources for downconverting or the infrared radiation sources, microwave sources, orradio frequency sources for up conversion) can be used to stimulate theluminescent particles in the encapsulated structures 10. In oneembodiment of the invention described here, the concentration ofluminescent particles in the medium or the spacing between theencapsulated structures 10 is set such that luminescent particles areseparated from each other in the medium by less than a UV depth ofpenetration into the medium. Higher concentrations are certainly usableand will generate higher UV fluxes should the energy source have enoughintensity to “light” all the luminescent particles.

For a relatively unclouded aqueous medium, UV-B irradiance decreases to1% after penetration into the water samples between 0.2 m and 1 m,whereas UV-A penetrates on the order of several meters. For suchmediums, the concentration of luminescent particles is more determinedby the time needed for the intended UV flux to produce deactivation oractivation of an agent in the medium, rather than having to be set basedon a concentration of luminescent particles where the medium itself doesnot occlude the UV stimulated emission from penetrating throughout themedium. The placement of the luminescent particles in the medium and inthe vicinity of the medium is not restricted by the optical density ofthe medium.

Accordingly, the upconverter structures of the invention (as discussedabove) can be provided on the interior of sealed quartz or glass tubesor can be provided coated on the surface of spheres or tubes, andfurther encapsulated with a silicate or another passivation layer. Inone embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone.

In this application, it is known that ultraviolet (UV) with a wavelengthof 254 nm tends to inactivate most types of microorganisms. Most juicesare opaque to UV due to the high-suspended solids in them and hence theconventional UV treatment, usually used for water treatment, cannot beused for treating juices. In order to make the process efficient, a thinfilm reactor constructed from glass has been used with the juice flowingalong the inner surface of a vertical glass tube as a thin film. See“Ultraviolet Treatment of Orange Juice” by Than et al. published inInnovative Food Science & Emerging Technologies (Volume 5, Issue 4,December 2004, Pages 495-502), the entire contents of which areincorporated herein by reference, Tran et al. reported that decimalreduction doses required for the reconstitute orange juices (OJ; 10.5°Brix) were 87-7 and 119±17 mJ/cm² for the standard aerobic plate count(APC) and yeast and moulds, respectively. They also reported that theshelf life of fresh squeezed orange juice was extended to 5 days with alimited exposure of UV (73.8 mJ/cm²). The effect of UV on theconcentration of Vitamin C was investigated using both HPLC andtitration methods of measurements. The degradation of Vitamin C was 17%under high UV exposure of 100 mJ/cm², which was similar to that usuallyfound in thermal sterilization. Enzyme pectin methylesterase (PME)activity, which is the major cause of cloud loss of juices, was alsomeasured. The energy required for UV treatment of orange juice (2.0 kWh/m³) was much smaller than that required in thermal treatment (82 kWh/m³). The color and pH of the juice were not significantly influencedby the treatment.

The invention described herein offers advantages over this approach inthat the upconverter structures of the invention can be placed insidefixtures such as quartz or glass (encapsulation structures) within theorange juice (or other fluid medium) and irradiated with NIR lightsupplied for example to the contained through manifold fiber optics toactivate the encapsulated upconverter structures of the invention in theorange juice.

While discussed with regard to orange juice, any other medium to besterilized including food products, medical products and cosmeticproducts could be treated using the technique of the invention describedherein.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Sterilization of Blood Products

U.S. Pat. No. 6,087,141 (the entire contents of which are incorporatedherein by reference) describes an ultraviolet light activated psoralenprocess for sterilization of blood transfusion products. The inventioncan be applied for the neutralization of AIDS and HIV or other viral orpathogenic agents in blood transfusion products. In this embodiment, atleast one photoactivatable agent is selected from psoralens, pyrenecholesteryloleate, acridine, porphyrin, fluorescein, rhodamine,16-diazorcortisone, ethidium, transition metal complexes of bleomycin,transition metal complexes of deglycobleomycin organoplatinum complexes,alloxazines, vitamin Ks, vitamin L, vitamin metabolites, vitaminprecursors, naphthoquinones, naphthalenes, naphthols and derivativesthereof having planar molecular conformations, porphorinporphyrins, dyesand phenothiazine derivatives, coumarins, quinolones, quinones,anthroquinones, porphycene, rubyrin, rosarin, hexaphyrin, sapphyrin,chlorophyl, chlorin, phthalocynine, porphyrazine, bacteriochlorophyl,pheophytin, texaphyrin macrocyclic-based component, or a metalatedderivative thereof. These photoactivatable agents serve as recipientsfor the secondarily generated light induced by the down conversion orupconversion.

The recipient in this and other embodiments of the invention can includeat least one of a laser dye, a fluorophore, a lumophore, or a phosphor.The laser dye can be at least one of p-terphenyl, sulforhodamine B,p-quaterphenyl, Rhodamine 101, curbostyryl 124, cresyl violetperchlorate, popop, DODC iodide, coumarin 120, sulforhodamine 101,coumarin 2, oxozine 4 perchlorate, coumarin 339, PCM, coumarin 1,oxazine 170 perchlorate, coumarin 138, nile blue A perchlorate,cournarin 106, oxatine 1 perchlorate, coumarin 102, pyridine 1, coumarin314T, styryl 7, coumarin 338, HIDC iodide, coumarin 151, PTPC iodide,coumarin 4, cryptocyanine, coumarin 314, DOTC iodide, coumarin 30, HITCiodide, coumarin 500, HITC perchlorate, coumarin 307, PTTC iodide,coumarin 334, DTTC perchlorate, coumarin 7, IR-144, coumarin 343, HDITCperchlorate, coumarin 337, IR-NO, coumarin 6, IR-132, coumarin 152,IR-125, coumarin 153, boron-dipyrromethere, HPTS, flourescein, rhodamine110, 2,7-dichlorofluorescein, rhodamine 65, and rhodamin 19 perchlorate,rhodamine b, and derivatives of these laser dyes that are modified byaddition the addition of appropriate substituents to modify solubilityor tune their interactions within the biological milieu.

In various embodiments of the invention, the recipients are secondaryagents performing other functions. Suitable secondary agents for theinvention include secondary emitters, cytotoxic agents, magneticresonance imaging (MRI) agents, positron emission tomography (PET)agents, radiological imaging agents, or photodynamic therapy (PDT)agents.

These photoactivatable agents (recipients and secondary agents) areintroduced into the blood product (or a patient's blood stream). NIRlight is applied to the blood product (or to the patient). Theupconverter structures of the invention (either included in the bloodproduct) or in encapsulated structures generate secondary light such asUV light which activates the photoactivatable agents in the bloodproducts. In one embodiment, the upconverter structures of the inventionare complexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone.

In a specific example, the photoactivatable agent is a psoralen, acoumarin, or a derivative thereof, and as discussed above, one cansterilize blood products in vivo (i.e., in a patient) or in a containerof the blood product (such as for example donated blood). The treatmentcan be applied to treat disorders such as for example a cancer cell, atumor cell, an autoimmune deficiency symptom virus, or a blood-bornegermicide is treated by the psoralen, the coumarin, or the derivativethereof.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Waste Water Detoxification

Photocatalysis has also been used as tertiary treatment for wastewaterto comply with regulatory discharge limits and to oxidize compounds thathave not been oxidized in the biological treatment. Photocatalysis hasbeen used to reduce or eliminate several pollutants (e.g., alkanes,alkenes, phenols, aromatics, pesticides) with great success. In manycases, total mineralization of the organic compounds has been observed.Several photocatalysts, such as CdS, Fe₂O₃, ZnO, WO₃, and ZnS, have beenstudied, but the best results have been achieved with TiO₂ P₂₅. Thesephotocatalyst can be used in the invention.

The wastewaters of an oil refinery are the waters resulting from washingthe equipment used in the process, undesirable wastes, and sanitarysewage. These effluents have high oil and grease contents, besides otherorganic compounds in solution. These pollutants form a residual chemicaloxygen demand (COD) that may pose serious toxic hazards to theenvironment.

It is known that photocatalysis can be used for waste water reductionremediation. U.S. Pat. No. 5,118,422 (the entire contents of which areincorporated herein by reference) to Cooper et al. describe anultraviolet driven photocatalytic post-treatment technique for purifyinga water feedstock containing an oxidizable contaminant compound. In thiswork, the water feedstock was mixed with photocatalytic semiconductorparticles (e.g., TiO₂, ZnO, CdS, CdSe, SnO₂, SrTiO₃, WO₃, Fe₂O₃, andTa₂O₅ particles) having a particle size in the range of about 0.01 toabout 1.0 micron and in an amount of between about 0.01% and about 0.2%by weight of the water. The water including the semiconductor mixture isexposed to band-gap photons for a time sufficient to affect an oxidationof the oxidizable contaminant to purify the water. Crossflow membranefiltration was used to separate the purified water from thesemiconductor particles. Cooper et al. show that the organic impuritycarbon content of simulated reclamation waters at nominal 40 PPM levelwere reduced to parts per billion using a recirculation batch reactor.

Cooper et al. identified that one important aspect of the photocatalyticprocess is the adsorption of the organic molecules onto the extremelylarge surface area presented by the finely divided powders dispersed inthe water. Cooper et al. further indicated that, in photoelectrochemicalapplications, advantage is taken of the fact that the solid phase (ametal oxide semiconductor) is also photo-active and that the generatedcharge carriers are directly involved in the organic oxidation. Theadsorption of the band-gap photon by the semiconductor particle resultsin the formation of an electron (e⁻)/hole (h⁺) pair. Cooper et al.explain that the electrons generated in the conduction band react withsolution oxygen forming the dioxygen anion (O²⁻) species whichsubsequently undergo further reactions resulting in the production ofthe powerfully oxidizing hydroxyl radical species, .OH. These powerfuloxidants are known to oxidize organic compounds by themselves.Additionally, Cooper et al. explain that the strongly oxidizing holesgenerated in the valence band have sufficient energy to oxidize allorganic bonds.

In the reactor of Cooper et al., turbulence is necessary in order toensure that the waste water contaminants and the photocatalytic titaniaparticles are exposed to the UV light. Cooper et al. explain that themost basic considerations of photocatalyst light adsorption and itsrelationship to convective mixing. For a 0.1 wt % photocatalyst loading,experiments have shown that 90% of the light is absorbed within 0.08 cm.This is primarily due to the large UV absorption coefficient of thephotocatalyst and therefore, most of the photoelectrochemistry occurswithin this illuminated region. By operating the reactor of Cooper etal. with a Reynolds number (Re) of 4000, a significant portion of thephotoactive region is ensured of being within the well mixed turbulentzone.

Santos et al. have reported in “Photocatalysis as a tertiary treatmentfor petroleum refinery wastewaters” published in Braz. J. Chem. Eng.vol. 23, No. 4, 2006 (the entire contents of which are incorporatedherein by reference), photocatalysis for tertiary treatment forpetroleum refinery wastewaters which satisfactorily reduced the amountof pollutants to the level of the regulatory discharge limits andoxidized persistent compounds that had not been oxidized in thebiological treatment. The treatment sequence used by the refinery(REDUC/PETROBRAS, a Brazilian oil refinery) is oil/water separationfollowed by a biological treatment. Although the process efficiency interms of biological oxygen demand (BOD) removal is high, a residual andpersistent COD and a phenol content remains. The refining capacity ofthe refinery is 41,000 m³/day, generating 1,100 m³/h of wastewater,which are discharged directly into the Guanabara Bay (Rio de Janeiro).Treating the residual and persistent COD remains a priority.

Santos et al. conducted a first set of experiments carried out in anopen 250 mL reactor containing 60 mL of wastewater. In the second set ofexperiments, a Pyrex® annular reactor containing 550 mL of wastewaterwas used (De Paoli and Rodrigues, 1978), as shown in FIG. 1. Thereaction mixtures inside the reactors were maintained in suspension bymagnetic stirring. In all experiments, air was continuously bubbledthrough the suspensions. A 250 W Phillips HPL-N medium pressure mercuryvapor lamp (with its outer bulb removed) was used as the UV-light source(radiant flux of 108 J·m⁻²·s⁻¹ at 8>254 nm). In one set of experiments,the lamp was positioned above the surface of the liquid at a fixedheight (12 cm). In the second set, the lamp was inserted into the well.All experiments by Santos et al. were performed at 25±1° C. The catalystconcentration ranged from 0.5 to 5.5 g L⁻¹ and the initial pH rangedfrom 3.5 to 9.

In one embodiment of the invention described herein, the upconverterstructures of the invention would be placed inside quartz or glassfixtures within the waste water or would be placed on silicaencapsulated structures within the waste water which, like thephotocatalytic TiO₂, could be entrained in the waste water during theirradiation.

Upon irradiation with for example NIR or IR radiation through forexample a manifold of fiber optics activation of the upconverterstructures of the invention would generate UV light in nearby presenceof the photocatalytic agent. In other words for this embodiment, theupconverter structures of the invention are mixed along with thephotocatalytic semiconductor particles in the waste water fluid stream,and the exterior activation energy source penetrates the container(e.g., a plastic or aluminum container) and irradiates the bulk of thewaste water, producing UV light throughout the waste water which in turndrives the photocatalytic reactions.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Photostimulation

Photostimulation is a field in which light is applied to in order toalter or change a physical property. For example, there has been anincreased focus on the use of biodegradable polymers in consumer andbiomedical fields. Polylactic acid (PLA) plastics andpolyhydroxyalkanoates (PHA) plastics have been playing a vital role infulfilling the objectives. But their relatively hydrophobic surfaceslimit their use in various applications. Hence, there is a need tosurface modify these film surfaces. Due to the lack of any modifiableside chain groups, workers have used a sequential two step photograftingtechnique for the surface modification of these biopolymers. In stepone, benzophenone was photografted on the film surface and in step two,hydrophilic monomers like acrylic acid and acrylamide werephotopolymerized from the film surfaces.

Workers have found that UV irradiation could realize an effective graftcopolymerization. UV-assisted photografting in ethanol has been used togrow hydrophilic polymers (e.g., poly(acrylic acid) and polyacrylamide)from the surfaces of PLA, PHA, and PLA/PHA blend films. In that work, afunctional polyurethane (PU) surface was prepared by photo-graftingN,N-dimethylaminoethyl methacrylate (DMAEM) onto the membrane surface.Grafting copolymerization was conducted by the combined use of thephoto-oxidation and irradiation grafting. PU membrane was photo-oxidizedto introduce the hydroperoxide groups onto the surface, then themembrane previously immersed in monomer solution was irradiated by UVlight. Results have shown prior to the invention that UV irradiation canrealize graft copolymerization effectively.

In the invention described herein, these processes are expedited by theinclusion of the upconverter structures of the invention in dispersionin the fluid medium being used for photostimulation. Upon NIRirradiation, the upconverter structures of the invention would generateUV light within the NIR penetration depth of the medium and permittingbatch or bulk type processing to occur in parallel inside the container.Further, when laser light is used for the NIR, the plastic surface canbe “written” onto such that inks would selectively absorb on thoseregions where surface of the polymer was exposed to the UV generatedlight.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Photodeactivation

In many industrial processes, especially food and beverage industries,yeasts are used to produce changes in a medium such as the conversion ofsugars in the raw product. One particularly prominent example is in thewine industry. Stopping the wine from fermenting any further wouldpreserve the current level of sweetness. Likewise, allowing the wine tocontinue fermenting further would only make the wine less sweet witheach passing day. Eventually the wine would become completely dry atwhich time the fermentation would stop on its own. This is becauseduring the fermentation process yeast turns the sugar into alcohol.

Wanting to stop the fermentation process is all good in and of itself.But unfortunately, there is really no practical way to successfully stopa fermentation dead in its tracks. Additives such as sulphite andsorbate can be added to stabilize a fermented product and stopadditional fermentation. Many winemakers will turn to sulfites such asthat found in Sodium Bisulfite or Campden tablets for the answer. But,these two items are not capable of reliably killing enough of the yeastto guarantee a complete stop of the activity—at least not at normaldoses that leave the wine still drinkable.

Once the bulk of the sulfites from either of these ingredients dissipatefrom the wine into the air—as sulfites do—there is a very strong chancethat the remaining few live yeast cells will start multiplying andfermenting again if given enough time. This usually happens at a mostinconvenient time, like after the wine has been bottled and stowed away.

Potassium sorbate is another ingredient that many winemakers considerwhen trying to stop a wine from fermenting any further. There is a lotof misunderstanding surrounding this product. It is typically called forby home wine making books when sweetening a wine. This is a situationwhere the fermentation has already completed and is ready for bottling.One adds the potassium sorbate along with the sugar that is added forsweetening.

The potassium sorbate stops the yeast from fermenting the newly addedsugar. So, many winemakers assume potassium sorbate can stop an activefermentation as well, but, potassium sorbate does not kill the yeast atall, but rather it makes the yeast sterile. In other words, it impairsthe yeast's ability to reproduce itself. But, it does not hinder theyeast's ability to ferment sugar into alcohol.

Ultraviolet light is known to destroy yeast cultures, but has restrictedapplications due to the inability of UV light to penetrate throughoutthe fluid medium. While heat can be used to destroy the yeast activity,cooking of the product may be premature or may produce undesirablechanges in the consistency and taste. For liquid or fluid food products,the same techniques described above could be used for the applicationdescribed here. For non-liquid products, energy modulation agents withlittle and preferably no toxicity (e.g. Fe oxides or titanium oxides)could be added. Here, the concentration of these additives would likelybe limited by any unexpected changes in taste.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Photoactivated Cross-Linking and Caring of Polymers

In this application, the upconverter structures of the invention areprovided and distributed into an uncured polymer based medium for theactivation of photosensitive agents in the medium to promotecross-linking and curing of the polymer based medium. In one embodiment,the upconverter structures of the invention are complexed with otherdown-converting luminescent particles or other energy modulation agentsprior to being added to the polymer.

For adhesive and surface coating applications, light activatedprocessing is limited due to the penetration depth of UV light into theprocessed medium. In light activated adhesive and surface coatingprocessing, the primary limitation is that the material to be cured mustsee the light—both in type (wavelength or spectral distribution) andintensity. This limitation has meant that one medium typically has totransmit the appropriate light. In adhesive and surface coatingapplications, any “shaded” area will require a secondary cure mechanism,increasing cure time over the non-shaded areas and further delaying curetime due to the existent of a sealed skin through which subsequentcuring must proceed.

Conventionally, moisture-curing mechanisms, heat-curing mechanisms, andphoto-initiated curing mechanisms are used to initiate cure, i.e.,cross-linking, of reactive compositions, such as reactive silicones,polymers, and adhesives. These mechanisms are based on eithercondensation reactions, whereby moisture hydrolyzes certain groups, oraddition reactions that can be initiated by a form of energy, such aselectromagnetic radiation or heat.

The invention described herein can use any of the following lightactivated curing polymers as well as others known in the art to whichthe upconverter structures of the invention are added.

For example, one suitable light activated polymer compound includes UVcuring silicones having methacrylate functional groups. U.S. Pat. No.4,675,346 to Lin, the disclosure of which is hereby expresslyincorporated herein by reference, is directed to UV curable siliconecompositions including at least 50% of a specific type of siliconeresin, at least 10% of a fumed silica filler and a photoinitiator, andcured compositions thereof. Other known UV curing silicone compositionssuitable for the invention include organopolysiloxane containing a(meth)acrylate functional group, a photosensitizer, and a solvent, whichcures to a hard film. Other known UV curing silicone compositionssuitable for the invention include compositions of an organopolysiloxanehaving an average of at least one acryloxy and/or methacryloxy group permolecule; a low molecular weight polyacrylyl crosslinking agent; and aphotosensitizer.

Loctite Corporation has designed and developed UV and UV/moisture dualcurable silicone compositions, which also demonstrate high resistance toflammability and combustibility, where the flame-retardant component isa combination of hydrated alumina and a member selected from the groupconsisting of organo ligand complexes of transition metals,organosiloxane ligand complexes of transition metals, and combinationsthereof. See U.S. Pat. Nos. 6,281,261 and 6,323,253 to Bennington. Theseformulations are also suitable for the invention.

Other known UV photoactivatable silicones include siliconesfunctionalized with, for example, carboxylate, maleate, cinnamate andcombinations thereof. These formulations are also suitable for theinvention. Other known UV photoactivatable silicones suitable for theinvention include benzoin ethers (“UV free radical generator”) and afree-radical polymerizable functional silicone polymers, as described inU.S. Pat. No. 6,051,625 whose content is incorporated herein byreference in its entirety. The UV free radical generator (i.e., thebenzoin ether) is contained at from 0.001 to 10 wt % based on the totalweight of the curable composition. Free radicals produced by irradiatingthe composition function as initiators of the polymerization reaction,and the free radical generator can be added in a catalytic quantityrelative to the polymerizable functionality in the subject composition.Further included in these silione resins can be silicon-bonded divalentoxygen atom compounds which can form a siloxane bond while the remainingoxygen in each case can be bonded to another silicon to form a siloxanebond, or can be bonded to methyl or ethyl to form an alkoxy group, orcan be bonded to hydrogen to form silanol. Such compounds can includetrimethylsilyl, dimethylsilyl, phenyldimethylsilyl, vinyldimethylsilyl,trifluoropropyldimethylsilyl, (4-vinylphenyl)dimethylsilyl,(vinylbenzyl)dimethylsilyl, and (vinylphenethyl)dimethylsilyl.

The photoinitiator component of the invention is not limited to thosefree radical generators given above, but may be any photoinitiator knownin the art, including the afore-mentioned benzoin and substitutedbenzoins (such as alkyl ester substituted benzoins), Michler's ketone,dialkoxyacetophenones, such as diethoxyacetophenone (“DEAP”),benzophenone and substituted benzophenones, acetophenone and substitutedacetophenones, and xanthone and substituted xanthones. Other desirablephotoinitiators include DEAP, benzoin methyl ether, benzoin ethyl ether,benzoin isopropyl ether, diethoxyxanthone, chloro-thio-xanthone,azo-bisisobutyronitrile, N-methyl diethanolaminebenzophenone, andmixtures thereof. Visible light initiators include camphoquinone,peroxyester initiators and non-fluorene-carboxylic acid peroxyesters.

Commercially available examples of photoinitiators suitable for theinvention include those from Vantico, Inc., Brewster, N.Y. under theIRGACURE and DAROCUR tradenames, specifically IRGACURE 184(1-hydroxycyclohexyl phenyl ketone), 907(2-methyl-1-[4-(methylthio)phenyl]-2-morpholino propan-1-one), 369(2-benzyl-2-N,N-dimethylamino-1-(4-morpholinophenyl)-1-butanone), 500(the combination of 1-hydroxy cyclohexyl phenyl ketone andbenzophenone), 651 (2,2-dimethoxy-2-phenyl acetophenone), 1700 (thecombination of bis(2,6-dimethoxybenzoyl-2,4,4-trimethyl pentyl)phosphine oxide and 2-hydroxy-2-methyl-1-phenyl-propan-1-one), and 819[bis(2,4,6-trimethyl benzoyl)phenyl phosphine oxide] and DAROCUR 1173(2-hydroxy-2-methyl-1-phenyl-1-propane) and 4265 (the combination of2,4,6-trimethylbenzoyldiphenyl-phosphine oxide and2-hydroxy-2-methyl-1-phenyl-propan-1-one); and IRGACURE 784DC(bis(.eta..sup.5-2,4-cyclopentadien-1-yl)-bis[2,6-difluoro-3-(1H-pyrrol-1-yl)phenyl]titanium).

Generally, the amount of photoinitiator (or free radical generators)should be in the range of about 0.1% to about 10% by weight, such asabout 2 to about 6% by weight. The free radical generator concentrationfor benzoin ether is generally from 0.01 to 5% based on the total weightof the curable composition.

A moisture cure catalyst can also be included in an amount effective tocure the composition. For example, from about 0.1 to about 5% by weight,such as about 0.25 to about 2.5% by weight, of the moisture curecatalyst can be used in the invention to facilitate the cure processbeyond that of photo-activated curing. Examples of such catalystsinclude organic compounds of titanium, tin, zirconium and combinationsthereof. Tetraisopropoxytitanate and tetrabutoxytitanate are suitable asmoisture cure catalyst. See also U.S. Pat. No. 4,111,890, the disclosureof which is expressly incorporated herein by reference.

Included in the conventional silicone composition (and other inorganicand organic adhesive polymers) suitable for the invention are variousinorganic fillers. For example, hollow microspheres supplied by Kishunder the trade name Q-CEL are free flowing powders, white in color.Generally, these borosilicate hollow microspheres are promoted asextenders in reactive resin systems, ordinarily to replace heavyfillers, such as calcium carbonate, thereby lowering the weight ofcomposite materials formed therewith. Q-CEL 5019 hollow microspheres areconstructed of a borosilicate, with a liquid displacement density of0.19 g/cm², a mean particle size of 70 microns, and a particle sizerange of 10-150 um. Other Q-CEL products are shown below in tabularform. Another commercially available hollow glass microsphere is sold byKish under the trade name SPHERICEL. SPHEREICEL 110P8 has a meanparticle size of about 11.7 microns, and a crush strength of greaterthan 10,000 psi. Yet other commercially available hollow glassmicrosphere are sold by the Schundler Company, Metuchen, N.J. under thePERLITE tradename, Whitehouse Scientific Ltd., Chester, UK and 3M,Minneapolis, Minn. under the SCOTCHLITE tradename.

In general, these inorganic filler components (and others such as fumedsilica) add structural properties to the cured composition, as well asconfers flowability properties to the composition in the uncured stateand increase the transmissivity for the UV cure radiation. When present,the fumed silica can be used at a level of up to about 50 weightpercent, with a range of about 4 to at least about 10 weight percent,being desirable. While the precise level of silica may vary depending onthe characteristics of the particular silica and the desired propertiesof the composition and the reaction product thereof, care should beexercised by those persons of ordinary skill in the art to allow for anappropriate level of transmissivity of the inventive compositions topermit a UV cure to occur.

Desirable hydrophobic silicas include hexamethyldisilazane-treatedsilicas, such as those commercially available from Wacker-Chemie,Adrian, Mich. under the trade designation HDK-2000. Others includepolydimethylsiloxane-treated silicas, such as those commerciallyavailable from Cabot Corporation under the trade designation CAB-O-SILN70-TS, or Degussa Corporation under the trade designation AEROSIL R202.Still other silicas include trialkoxyalkyl silane-treated silicas, suchas the trimethoxyoctyl silane-treated silica commercially available fromDegussa under the trade designation AEROSIL R805; and 3-dimethyldichlorosilane-treated silicas commercially available from Degussa underthe trade designation R972, R974 and R976.

While these inorganic fillers have extended the use of conventional UVcured to silicone systems to permit the curing of materials beyond askin depth of UV penetration, these inorganic fillers alone do notovercome shadowing effects and suffer from UV scattering whicheffectively makes for a smaller penetration depth. In the inventiondescribed herein, the inclusion of these inorganic fillers along withluminescing particles provide a mechanism by which uniform lightactivated cures can occur deep inside of the body of adhesive-solidifiedassemblies in regions that would normally be shadowed or not with thereach of external UV or other light sources.

Accordingly, in this example of the invention described herein,conventional silicone and polymeric adhesive or release or coatingcompositions are prepared using conventional mixing, heating, andincubation techniques. Included in these conventional compositions arethe upconverter structures of the invention. These compositions can thenbe applied to surfaces of objects to be fixed together or to surfaceswhere a hard coating is desired or cast in a curable form for theproduction of molded objects. These compositions upon activation willproduce radiant light for photoactivated cure of the luminescingparticle containing polymer composition. The density of the upconverterstructures in these compositions will depend on the “light transparency”of the luminescing particle containing composition. Where thesecompositions contain a significant amount of the inorganic filler asdiscussed above, the concentration of the upconverter structures can bereduced for example as compared to a composition with a black colorpigment where the light transparency will be significantly reduced.

U.S. Pat. No. 7,294,656 to Bach et al., the entire disclosure of whichis incorporated herein by reference, describes a non-aqueous compositioncurable by UV radiation broadly containing a mixture of two UV curableurethane acrylates that have several advantages over conventionalradiation-curable compositions. The Bache et al. compositions can becured in a relatively short time using UV-C (200-280 nm), UV-B (280-320nm), UV-A (320-400 nm) and visible (400 nm and above) radiation. Inparticular, Bache et al. compositions can be cured using radiationhaving a wavelength of 320 nm or more. When fully cured (regardless ofthe type of radiation used), the Bach et al. compositions exhibithardnesses and impact resistances at least comparable to conventionalcoatings.

In the invention described here, the upconverter structures are added tothese Bach et al. compositions. Due to the fact that the exterior energysource penetrates deeper into the entirety of the Bach et al.compositions, thicker surface coatings can be realized. Further, thecoatings can be applied to intricate surfaces having for example beenprepared with recesses or protrusions.

In one embodiment, the upconverter structures of the invention arecomplexed with the X-ray down converting particles or other energymodulation agents permitting for example X-ray irradiation to alsoassist in this process. In one embodiment, the X-ray down convertingparticles or other energy modulation agents or metallic structuresdescribed herein permit X-ray irradiation to be used alone or incombination with the up converting particles.

Generalized Upconversion

The invention as described above can be viewed for its aspects ofexposing an agent to one source of light or radiation (an initiationsource) of a relatively low energy and having the agent produce light orradiation at a relatively higher energy. In one embodiment of theinvention, a change is produced in a medium. The change is produced by(1) placing in a vicinity of the medium a nanoparticle or an otherwiseupconverting structure, and (2) applying the initiation energy from anenergy source through the artificial container to the medium, whereinthe emitted light directly or indirectly produces the change in themedium.

The nanoparticle or the otherwise upconverting structure in oneembodiment is configured, upon exposure to a first wavelength λ₁ ofradiation, to generate a second wavelength λ₂ of radiation having ahigher energy than the first wavelength λ₁. The nanoparticle or theotherwise upconverting structure in one embodiment includes a metallic Tmetallic structure disposed in relation to the nanoparticle (e.g. ametallic shell covering a fraction of the nanoparticle) A receptor inthe medium, upon activation by the second wavelength λ₂, generatesdirectly or indirectly a photostimulated change in the medium. In oneembodiment of the invention, a physical characteristic of metallicstructure (such as those described above) is set to a value where asurface plasmon resonance in the metallic structure resonates at afrequency which provides spectral overlap with either the firstwavelength λ₁ or the second wavelength λ₂.

The metallic structure in one embodiment has a radial dimension of themetallic shell set to a value where a surface plasmon resonance in themetallic shell resonates at a frequency which provides spectral overlapwith either the first wavelength t, or the second wavelength λ₂. Thenanoparticle or the otherwise upconverting structure in one embodimentis configured to emit light into the medium upon interaction with aninitiation energy having energy in the range of λ₁.

The change produced in the medium can cure a radiation-curable medium byactivating a photoinitiator in the radiation-curable medium. The changeproduced can result in a photo-stimulated change to a medium. The changeproduced can result in a radiation cured medium. The change produced canresult in a sterilized medium. The change produced can activate atherapeutic drug.

The agents in one embodiment of the invention can include not only theupconverter nanoparticles discussed above, but also can include theinfrared-triggered phosphors discussed above. Furthermore, the agentscan include fluorescent molecules or luminescent inorganic molecules orphosphorescent molecules (acting as either down or up converters invarious embodiments). Suitable agents include, but are not limited to, ametal nanoparticle or a biocompatible metal nanoparticle, a metal coatedor uncoated with a biocompatible outer layer, a chemiluminescentmolecule whose rate of luminescence is increased by microwaveactivation, fluorescing dye molecule, gold nanoparticle, a water solublequantum dot encapsulated by polyamidoamine dendrimers, a luciferase, abiocompatible phosphorescent molecule, a biocompatible fluorescentmolecule, a biocompatible scattering molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelatecapable of intense luminescence. Multiple types of agents can beincluded in the medium.

For many of these agents, the initiation source may well be lowfrequency sources such as microwave or radio frequency irradiation,where in one embodiment of the invention localized heating of the agentenhances generation of a secondary light and in another embodimentlocalized field enhancements from the microwave field present in themedium enhance fluorescence, as described in “Microwave-AcceleratedMetal-Enhanced Fluorescence (Mamef) With Silver Colloids in 96-WellPlates: Application to Ultra Fast and Sensitive Immunoassays, HighThroughput Screening and Drug Discovery,” by Asian et al in Journal ofImmunological Methods 312 (2006) 137-147.

For many of these agents, the initiation source may well be lowfrequency sources such as microwave or radio frequency radiation, wherein one embodiment of the invention absorption of the microwave radiationby upconverters results in subsequent emission at higher energies towardthe infrared, visible, and ultraviolet. The degree to which theupconverted radiation is applicable to the applications described abovewill be dependent on the conversion efficiencies of the specific metalshell/dielectric core nanostructures and will be dependent on theefficiency of a recipient molecule linked to the specific metalshell/dielectric core nanostructures to absorb the upconverted light.

In one embodiment, there is provided a system for energy upconversion.The system includes a nanoparticle configured in such a way that uponexposure to a first set of radiation having a wavelength λ₁ or centeredaround wavelength λ₁ (also known as a frequency window centered aroundfrequency f1 or v₁), to generate a second set of radiation centeredaround wavelength λ₂ having a higher quantum energy level than the firstset of radiation centered around or having wavelength λ₁. The system caninclude for example a metallic shell encapsulating at least a fractionof the nanoparticle. The radial dimension of the metallic shell is setto within a range of suitable values where surface plasmon resonance cantake place in the metallic shell under the impingement or incidence ofthe first set of operating frequencies of interest; this is accomplishedthrough a spectral overlap of the operating frequencies with either thefirst set of radiation having wavelengths centered at λ₁ or the secondradiations centered around wavelength λ₂. The range of frequencies in afrequency window centered on a desirable center frequency can be verynarrow and under ideal conditions the frequency window contains only onemonochromatic radiation having a single frequency.

The system can include for example a metallic structure disposed inrelation to the nanoparticle where a physical characteristic of metallicstructure (such as those described above) is set to a value where asurface plasmon resonance in the metallic structure resonates at afrequency which provides spectral overlap of the operating frequencieswith either the first set of radiation having wavelengths centered at λ₁and/or the second radiations centered around wavelength λ₂. The range offrequencies in a frequency window centered on a desirable centerfrequency can be very narrow and under ideal conditions the frequencywindow contains only one monochromatic radiation having a singlefrequency.

In one embodiment of the invention, the surface plasmon resonanceincreases an intensity of at least one of the first wavelength λ₁ or thesecond wavelength λ₂ in a vicinity of the nanoparticle, to therebyenhance the likelihood that the desirable reaction takes place.

In another embodiment, there is provided a system for producing aphotostimulated reaction in a medium. The system includes a nanoparticleconfigured, upon exposure to a first radiation having wavelength λ₁, togenerate a second radiation having wavelength λ₂ with a higher quantumenergy level than the first radiation having wavelength λ₁. The systemincludes a metallic structure disposed in relation to the nanoparticle(e.g., a metallic shell encapsulating at least a fraction of thenanoparticle) and includes a receptor disposed in the medium inproximity to the nanoparticle. The receptor upon activation by thesecond wavelength λ₂ generates the photostimulated reaction.

In yet another embodiment, there is provided a nanoparticle structureincluding a sub 1000 nm dielectric core and a metallic shellencapsulating at least a fraction of the nanoparticle. The dielectriccore includes at least one of Y₂O₃, Y₂O₂S, NaYF₄, NaYbF₄, YAG, YAP,Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, Na-doped YbF₃,or SiO₂. These dielectric cores can be doped with Er, Eu, Yb, Tm, Nd,Tb, Ce, Y, U, Pr, La, Gd and other rare-earth species or a combinationthereof. Such nanoparticle structures including one or more of thesedielectric cores can exhibit in certain embodiments surface plasmonresonance in the metallic shell to enhance up conversion of light orelectromagnetic radiation from a first wavelength λ₁ to a secondwavelength λ₂.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Obviously, additional modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

The invention claimed is:
 1. A method for modifying a target structurewhich mediates or is associated with a biological activity, comprising:placing in a vicinity of a target structure in a subject in need oftreatment an agent receptive to microwave radiation or radiofrequencyradiation; and applying as an initiation energy said microwave radiationor radiofrequency radiation by which the agent directly or indirectlygenerates emitted light in the infrared, visible, or ultraviolet range,wherein the emitted light contacts the target structure and induces apredetermined change in said target structure in situ, wherein saidpredetermined change modifies the target structure and modulates thebiological activity of the target structure.
 2. The method of claim 1,wherein placing an agent comprises placing a nanoparticle including ametallic structure in relation to the nanoparticle.
 3. The method ofclaim 2, wherein placing the nanoparticle comprises placing ananoparticle having a dielectric core.
 4. The method of claim 3, whereinplacing the nanoparticle comprises at least one of: placing ananoparticle having a metallic shell including at least one of aspherical shell, an oblate shell, a crescent shell, a multilayer shell,or an array of metal nanoislands on the surface of the core nanoparticlein the vicinity of the target structure; placing a nanoparticle havingan array of metal nanoislands on the surface of the core nanoparticle inthe vicinity of the target structure; or placing a nanoparticle havingan array of nanoislands on the surface of the core nanoparticle in thevicinity of the target structure.
 5. The method of claim 3, wherein thenanoparticle comprises a metallic structure comprising at least one ofelement selected from the group consisting of Au, Ag, Cu, Ni, Pt, Pd,Co, Ru, Rh, Al, Ga, and a combination thereof.
 6. The method of claim 3,wherein the nanoparticle comprises a core comprising least one compoundselected from the group consisting of Y₂O₃, Y₂O₂S, NaYF₄, YAG, YAP,Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃, Na-doped YbF₃,SiO₂ and alloys or layers thereof.
 7. The method of claim 3, wherein thenanoparticle comprises a dopant comprising at least one of elementselected from the group consisting of Er, Eu, Yb, Tm, Nd, Tb, Ce, Y, U,Pr, La, Gd, other rare-earth species and a combination thereof.
 8. Themethod of claim 1, wherein placing an agent comprises placing at leastone of a phosphorescent molecule, a fluorescent molecule, or aluminescent inorganic molecule, each displaying emission upon exposureto said microwave radiation or radiofrequency radiation.
 9. The methodof claim 1, wherein placing an agent comprises placing at least one of achemiluminescent molecule, displaying emission upon exposure to saidmicrowave radiation or radiofrequency radiation.